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Adapting the Smart-seq2 Protocol for Robust Single Worm RNA-seq
改编Smart-seq2实验方案以实现稳定的单蠕虫RNA测序   

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参见作者原研究论文

本实验方案简略版
PLOS Pathogens
Apr 2017

Abstract

Most nematodes are small worms that lack enough RNA for regular RNA-seq protocols without pooling hundred to thousand of individuals. We have adapted the Smart-seq2 protocol in order to sequence the transcriptome of an individual worm. While developed for individual Steinernema carpocapsae and Caenorhabditis elegans larvae as well as embryos, the protocol should be adaptable for other nematode species and small invertebrates. In addition, we describe how to analyze the RNA-seq results using the Galaxy online environment. We expect that this method will be useful for the studying gene expression variances of individual nematodes in wild type and mutant backgrounds.

Keywords: RNA-seq (RNA-seq), Transcriptome (转录), C. elegans (秀丽隐杆线虫), S. carpocapsae (斯氏线虫)

Background

Low input RNA-seq protocols and amplification kits, such as Smart-seq (Takara Bio, USA, Inc) and SuperAmp (Miltenyl Biotec, Inc), have been increasingly developed and commercialized as a response to the growing prevalence of low input RNA-seq studies based on small tissues, single microorganisms, and single cells. These studies often explore and address heterogeneous gene expression among individuals of a certain population, such as a population of cells, a complex tissue, or a population of microscopic organisms. Improvements and adaptations of low input RNA-seq protocols for microscopic organisms, such as nematodes, will greatly benefit the field of nematology by allowing for the analysis of gene expression heterogeneity at the single nematode level. Here we have adapted the single cell RNA-seq protocol, Smart-seq2 (Picelli et al., 2013 and 2014; Trombetta et al., 2014), for single nematode RNA-sequencing. We successfully utilized adapted versions of this protocol in the transcriptomic analysis of the insect-parasitic nematode, Steinernema carpocapsae (Lu et al., 2017) as well as in the analysis of individual embryos and L1 larvae from two Steinernema and two Caenorhabditis species including C. elegans (Macchietto et al., 2017), but this protocol can be adapted for any species of nematode. While this protocol will work on nematodes without already sequenced genomes or transcriptomes, we limit our computational analysis to organisms with published genome annotations, such as S. carpocapsae (Dillman et al., 2015). Our need for single nematode RNA-sequencing arose as a method to circumvent the limitations of working with samples with low-inputs of RNA. For example, many of our in vivo experiments limited the number of nematodes we could utilize. Single nematode RNA-seq has allowed us to efficiently obtain high resolution gene expression data from these nematodes. The protocol has also enabled us to collect individual embryos to map out time courses of nematode embryonic development for comparative transcriptomics across multiple species. The development and advancement of low input RNA-seq protocols will aid investigators in circumventing issues related to using individual organisms and specialized/limited samples.

Materials and Reagents

  1. Gloves
  2. 8-strip, nuclease-free, 0.2-ml, thin-walled PCR tubes with caps (SARSTEDT, catalog numbers: 72.985.002 and 65.989.002 )
  3. Needle 25 G 1.5 inch regular (BD, PrecisionGlide, catalog number: 305127 )
  4. QubitTM assay tubes (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q32856 )
  5. Pipette tips
  6. 1.5 ml Eppendorf tube
  7. Spatulas
  8. 70% ethanol or RNase away
  9. Proteinase K (QIAGEN, catalog number: 19131 )
  10. RNasin ribonuclease inhibitor (RNase inhibitor) (Promega, catalog number: N2611 )
  11. UltraPure DNase/RNase free distilled water (Thermo Fisher Scientific, GibcoTM, catalog number: 10977015 )
  12. Oligo-dT30VN primer (ordered from IDT (https://www.idtdna.com/site)): 5’-AAGCAGTGGTATCAACGCAGAGTACT30VN-3’
    Note: This oligonucleotide anneals to all the RNAs containing a poly(A) tail. The 3’ end of this oligonucleotide contains ‘VN’, where ‘N’ is any base and ‘V’ is either A, C or G. The two terminal nucleotides are necessary for anchoring the oligonucleotide to the beginning of the poly(A) tail and for avoiding unnecessary amplification of long stretches of adenosines. Dissolve the oligonucleotide in TE buffer to a final concentration of 100 μM. Store this oligo at -20 °C for 6 months.
  13. dNTP mix (10 mM each) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0192 )
  14. Superscript II reverse transcriptase kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18064014 )
  15. LNA-modified TSO (ordered from Exiqon (http://www.exiqon.com/))
    5’-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3’
    Note: At the 5’ end, this TSO carries a common primer sequence, whereas, at the 3’ end, there are two riboguanosines (rG) and one LNA-modified guanosine (+G) to facilitate template switching. TSO dissolved in TE buffer can be stored in 100 μM aliquots at -80 °C for 6 months. Avoid repeated freeze-thaw cycles.
  16. Betaine (BioUltra ≥ 99.0%) (Sigma-Aldrich, catalog number: 61962 )
  17. Magnesium chloride (MgCl2; anhydrous) (Sigma-Aldrich, catalog number: M8266 )
  18. Kapa HiFi HotStart ReadyMix (Kapa Biosystems, catalog number: KK2602 )
  19. IS PCR oligo (ordered from IDT (https://www.idtdna.com/site))
    5’-AAGCAGTGGTATCAACGCAGAGT-3’
    Note: This oligonucleotide acts as PCR primer in the amplification step after RT. Dissolve the oligonucleotide in TE buffer to a final concentration of 100 μM. This oligo can be stored at -20 °C for 6 months.
  20. Agencourt Ampure XP beads (Beckman Coulter, catalog number: A63881 )
  21. Ethanol 99.5% (vol/vol) (Kemethyl, catalog number: SN366915-06 )
  22. QubitTM dsDNA HS assay kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: Q32854 )
  23. Agilent high sensitivity DNA kit (Agilent Technologies, catalog number: 5067-4626 )
  24. Buffer PM (QIAGEN, catalog number: 19083 )
  25. Nextera DNA library prep kit (24 samples) (Illumina, catalog number: FC-121-1030 )
  26. QIAquick PCR purification kit (50) (QIAGEN, catalog number: 28104 )
  27. Phusion high fidelity PCR master mix with HF buffer, 500 reactions (New England Biolabs, catalog number: M0531L )
  28. Tris-HCl pH 8.0 (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9850G )
  29. Triton X-100 (Sigma-Aldrich, catalog number: T9284 )
  30. EDTA pH 8.0 (Mediatech, catalog number: 46-034-Cl )
  31. Polysorbate 20, Acros OrganicsTM (Tween 20) (Acros Organics, catalog number: 233362500 )
  32. RNaseZap (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9780 )
  33. List of sequencing indexes (Buenrostro et al., 2015) (see Supplemental file)

Equipment

  1. Pipettes
  2. Mini-centrifuge with head for 8-strip PCR tubes
  3. Vortexer
  4. Thermocycler
  5. Stereo microscope
  6. Qubit® Fluorometer
  7. Agilent 2100 Bioanalyzer (Agilent Technologies, model: Agilent 2100 , catalog number: G2938C)
  8. Magnetic stand 96 (Thermo Fisher Scientific, catalog number: AM10027 )

Procedure

Warning: RNA is easily degradable by RNases and not very stable on ice! Therefore, clean all surfaces with 70% ethanol or RNase away, change gloves often and sterilize all materials used. Lysed samples should not be kept on ice for more than one hour as mRNA will degrade.
Notes:

  1. This protocol can be used for all larval stages, dauers and embryos of nematodes.
  2. A new sterile needle should be used for each worm.


  1. Isolating and lysing nematodes from cultured plates
    Note: Prepare the buffers freshly on the day of worm collection.
    1. Prepare lysis buffer (see Recipes in the end of the protocol) with Proteinase K by combining 46.8 µl of lysis buffer stock with 3.2 µl of Proteinase K for a total of 50 µl.
    2. From Step A1 combine 18 µl of lysis buffer with Proteinase K with 2 µl RNase inhibitor for a total of 20 µl of lysis buffer.
    3. Use the lysis buffer with Proteinase K and RNasin ribonuclease inhibitor for lysing worms.
    4. Set up a station for nematode collection (Figure 1A).
    5. Wash nematodes three times with deionized water:
      1. To wash nematodes from cultured plates, add 1 ml of water to plate.
      2. Swirl the plate three times to dislodge nematodes.
      3. Transfer worms with a pipette to a 1.5 ml Eppendorf tube.
      4. Briefly spin down.
      5. Remove supernatant and bring up to 1 ml with water.
      6. Repeat Steps A5d and A5e two more times or until the water becomes clear.
    6. Collect one worm using a pipette in 2 µl of water and transfer to the wall of PCR tube (Figure 1B). Cut the worm with 25 gauge needle (Figures 1C-1D) (Video 1).
      Note: Cutting is suggested for all larval stages. Note that we did not cut the embryos.

      Video 1. A video demonstrating the procedure for cutting a single nematode in preparation for RNA extraction

    7. Add 2 µl lysis buffer with Proteinase K and RNasin ribonuclease inhibitor.
    8. Flick tube to mix, and spin down tubes in a mini-centrifuge.
    9. Incubate samples on the thermocycler using the following program on PCR cycler.
      1. 65 °C–10 min
      2. 85 °C–1 min
      3. 4 °C
    10. Transfer samples to ice.
    11. Add 1 µl oligo-dT VN primer (10 μM) and 1 µl dNTP mix to each tube. Flick tubes to mix, and spin down tubes.
    12. Incubate samples on the thermocycler at 72 °C for 3 min.
    13. Take samples from the thermocycler and immediately place them on ice.


      Figure 1. Preparing and cutting an individual nematode for lysis. A. A picture of the setup used to isolate, cut, and prepare individual nematodes for RNA-seq. B. A picture of an individual nematode isolated in a pipette tip, being transferred to a PCR tube to be cut. C. A picture of a single nematode in 2 µl of DEPC-treated water. D. A picture of a single nematode in a PCR tube that has been cut in half using a 25 gauge needle. In panels B-D the individual nematode is circled in red.

  2. Smart-seq2 reverse transcription (RT) of mRNA
    1. Prepare reverse transcription master mix (RT MM). (No DNase required.)


    2. Add 5.7 μl of RT MM to each PCR tube.
    3. Quickly vortex and spin down in a mini-centrifuge.
    4. Put on thermocycler and run the following program:

      Note: Stop point. Store samples -20 °C.

  3. Smart-seq2 PCR amplification (Figure 2)
    1. Place samples on ice. Prepare KAPA HiFi master mix.


    2. Add 15 μl of KAPA HiFi master mix to each sample.
    3. Put on thermocycler and run the following program:

      Note: Stopping point. Samples can be stored at -20 °C indefinitely.


      Figure 2. Overview of the Smart-seq2 protocol adapted for individual nematodes. Modified from Picelli et al. (2014). 1. A nematode is cut and lysed to release total RNA. 2. The Oligo(dT) binds the poly(A) tail at the 3’ end of mRNA (section A of Procedure). 3. The 1st strand of cDNA is synthesized by MMLV reverse transcriptase (RT) which adds non-template guided cytosines at the 3’ end of the cDNA. These cytosines are used to anchor the LNA-modified TSO. Reverse transcriptase then uses the TSO as a template to complete the 1st strand (section B of Procedure). 4. The ISPCR primer anneals to the 3’ end of the 1st strand allowing DNA polymerase to bind and to synthesize the 2nd strand of cDNA and subsequently amplify the cDNA (section C of Procedure). 5. The double-stranded (ds) cDNA is purified and checked for quality (sections D and E of Procedure). 6. Transposomes tagment the ds cDNA by fragmenting it and ligating adapter sequences (section F of Procedure). 7. The tagmented ds cDNA is cleaned and 8. Ligated to sample specific indexes (section G of Procedure). 

  4. cDNA bead clean-up

    1. Take samples out of -20 °C and let them sit at room temperature for 10 min.
    2. Take Ampure XP beads from fridge and vortex at high speed for 1 min. The solution should be homogenous.
    3. Aliquot 26 μl of Ampure XP beads into a 1.5 ml Eppendorf tube. Beads need to be warmed up to room temperature approximately 8 min.
    4. Add the sample (~26 μl) to the Ampure XP beads (1:1). Pipette up and down 10 times until thoroughly mixed.
    5. Incubate the sample with the Ampure XP beads for 8 min at room temperature on a tube rack.
    6. Make 600 μl of fresh 80% nuclease-free ethanol.
    7. After incubation, place sample on a magnetic bead stand for 5 min.
      Note: The beads will be bound to cDNA of interest.
    8. Remove supernatant using pipette without disturbing the beads.
      Note: Place the supernatant into the old sample PCR tube and keep it until the cDNA concentration is checked.
    9. Leave tubes on magnetic bead stand, add 200 μl of 80% ethanol to the beads. Wait 30 sec.
    10. Remove supernatant using a needle attached to a vacuum line or a pipette, being careful not to disturb the beads.
      Note: Do not let beads dry out.
    11. Repeat Steps D9-D10 two more times.
    12. Let beads dry on the magnetic stand for ~5 min with the cap open. Monitor the drying.
    13. When the beads begin to crack, remove the tube from the magnet and immediately add 17.5 μl of EB buffer to the beads. Pipette up and down 10 times to thoroughly mix. The solution should be homogenous and brown.
    14. Incubate the sample on a tube rack at room temperature for 2 min.
    15. Put the sample back on the magnetic bead stand for 2 min or until the liquid turns clear. Collect 15 μl of supernatant from the tube while on the magnetic stand. Be careful not to disturb beads. Place the supernatant into a clean labeled Eppendorf tube and place on ice.
      Note: There should be no brown specs in the liquid collected. If you aspirate any beads, return the sample to the bead tube and repeat Steps D14-D15.
    16. Discard tubes left in the magnetic stand.

  5. Measurement of cDNA concentration and sample quality check
    Note: Calibrate the Qubit Fluorometer according to manufacturer’s guide.
    1. Qubit reagents for one sample:
      198 μl of dsDNA HS Buffer
      1 μl of dsDNA HS reagent 200x
      1 μl of cDNA
      Add the dsDNA HS reagent, dsDNA HS buffer and cDNA to a Qubit tube. Quickly vortex and spin down in a mini-centrifuge.
    2. Incubate the tube for 2 min in the dark.
    3. Place cDNA sample into the Qubit fluorometer. On the Qubit screen, select ‘DNA’, select ‘High Sensitivity’, and select ‘read sample’. Select ‘read stock concentration’ and change the volume to ‘1 μl’. Record the cDNA concentration (ng/μl).
    4. Run BioAnalyzer (BA) on the cDNA following the protocol for the Agilent High Sensitivity DNA Kit.
      Samples should be run on a BioAnalyzer machine using an Agilent High Sensitivity DNA Kit to generate profiles of the distribution of cDNA lengths (representative of the mRNA transcript lengths) in each sample. If the sample is not degraded, you will see a clear peak in the fragment sizes in the 500-10,000 bp range with no peaks at smaller fragment sizes indicative of full length cDNA resulting from full length mRNA transcripts (Figure 3A). The profile will show no peaks or many small peaks if the mRNA transcripts were degraded (likely due to RNase contamination or mishandling of the RNA) (Figure 3B).


      Figure 3. Examples of Smart-seq2 BioAnalyzer (BA) profiles. A. An ideal BA profile of cDNA showing full length transcripts with large peaks around 1,000 bp and no PCR primer contamination (70 bp). B. A BA profile of cDNA made from highly degraded RNA resulting in an abundance of small fragments shown by multiple peaks between 35 and 300 bp.

  6. Tagmentation of cDNA (Figure 2)
    Notes:
    1. All reagents and samples should be kept on ice.
    2. This protocol uses the standard Nextera DNA library prep kit rather than Nextera XT DNA sample prep kit.
    3. Buffer PM is used because it has acetic acid and consequentially addresses the pH value of samples.

    1. Set a heating block to 55 °C and warm 30 μl of EB buffer.
    2. Calculate how to get 20 ng of cDNA from sample in 8 μl (see example below).


    3. In a PCR tube place 20 ng of Ampure-purified PCR product. Add EB to bring volume to 8 μl.
    4. In a NEW PCR tube, add 10 μl of Tagment DNA buffer from Nextera DNA Library Prep Kit.
    5. Add 2.2 μl Tagment DNA enzyme 1 from Nextera DNA Library prep Kit.
    6. Set a new P20 pipette to 15 μl and mix the transposase enzyme and transposase buffer by pipetting up and down 10 times.
    7. Transfer all of the mixed transposase enzyme and buffer into your cDNA and IMMEDIATELY pipette up and down at least 10 times to mix very well.
    8. Place sample on the PCR block for 5 min at 55 °C to initiate the tagmentation reaction.
    9. While sample is on PCR block, obtain a QIAquick DNA cleanup column, buffer PM and PE. The tagmented cDNA will be passed through this column to remove the enzyme from the DNA fragments.
    10. Add 60 μl of buffer PM to the 20 μl of tagmented cDNA. Mix well by pipetting up and down until the solution becomes clear.
    11. Pipette the entire volume into the QIAGEN spin column.
    12. Spin the QIAGEN column down in a centrifuge at 12,470 x g for 1 min. The cDNA will stick to the white filter in the column, and the liquid will pass through into the collection tube.
    13. Dump the flow-through into the trash. Place the column back inside the collection tube.
    14. Add 750 μl of buffer PE to the column.
    15. Spin the QIAGEN column down in a centrifuge at 12,470 x g for 1 min.
    16. Dump the flow-through into the trash. Place the column back inside the collection tube.
    17. Spin the QIAGEN column down in a centrifuge at 12,470 x g for 1 min.
    18. Place the column into a new, clean the Eppendorf tube that is labelled with the sample name.
    19. Add 30 μl of warmed EB buffer to the center of the white filter inside the column, being careful to not touch the filter with the pipette tip or to pipette the buffer onto the walls of the column.
    20. Let the column sit for 1 min.
    21. Spin down the column in a centrifuge at 12,470 x g for 1 min.
    22. Remove the column and keep the flow-through.
    23. The flow-through is the tagmented cDNA.
      Note: At this step samples can be handed to the facility at which they will be sequenced. If barcodes are to be added by researcher, follow the next steps.

  7. Amplification of adapter-ligated fragments (Figure 2)
    1. In a PCR tube add the following reagents:


    2. Put samples on PCR cycler and run the following program (Buenrostro et al., 2015):

      Note: After the stopping point, samples can be stored at -20 °C indefinitely.

  8. DNA library bead clean-up

    Follow steps described in Procedure D with the following changes:
    1. Step D3, use 70 μl of Ampure XP beads (1:1).
    2. Step D4, add the sample (~70 μl).
    3. Step D14, add 30 μl of EB.
    4. Step D16, collect 27.5 μl of supernatant.

  9. Measurement of DNA library concentration and sample quality check
    1. Follow steps in Procedure E to acquire DNA library concentration with Qubit.
    2. Run BioAnalyzer to check quality of library. (Optional, but highly recommended!)
      Samples should be run on a BioAnalyzer to check for quality of tagmentation from Procedure F. A fully tagmented library will have a BioAnalyzer profile with a clear peak around 200 bp (Figure 4A). A partially tagmented library will have a BioAnalyzer profile with a uniform plateau between 150 to 1,000 bp and possibly some smaller peaks below 150 bp or above 1,000 bp (Figure 4B).


      Figure 4. Examples of Smart-seq2 BioAnalyzer profiles after tagmentation and library preparation. A. An example of a sample that has been fully tagmented shows one peak around 200 bp. B. An example of a sample that was not fully tagmented shows a broad distribution of fragments ranging from 150-1,000 bp.

  10. Library sequencing
    The libraries can be handed to a sequencing facility. Samples are sequenced using pair-end, 43 base pair reads. Pair-end sequencing improves the quality of the dataset enabling reads to more accurately align to the reference genome. Libraries can also be sequenced with single-end reads. Each sample should be sequenced to a depth of at least 10 million reads to reliably detect one transcript per million (TPM).

Data analysis

Analysis of Smart-seq2 data
RNA-seq libraries are typically sequenced to a depth of 10-20 million reads to reliably profile gene expression levels in samples of interest. Although fast and reliable software such as STAR (Dobin et al., 2013), Kallisto (Bray et al., 2016), and Salmon (Patro et al., 2017) have been developed for analyzing reads and obtaining gene/isoform expression levels, analysis of high throughput data usually requires computing and data storage resources beyond the capabilities of a typical desktop or laptop machine. Such tasks require access to a high performance computing cluster or online resources such as Galaxy (Afgan et al., 2016). Galaxy provides free access to analysis tools and computing/storage resources through a user-friendly online interface. Sequencing data generated using any of the various sequencing protocols can be uploaded to Galaxy that has appropriate analysis tools available to obtain interpretable results. Here we present a detailed tutorial for uploading Steinernema carpocapsae RNA-seq data to Galaxy and obtaining gene expression levels using Salmon.

  1. Creating a user account
    Use of Galaxy resources requires a user account. A free user account can be obtained by registering at Galaxy. Log on to usegalaxy.org using any web browser of choice.
    1. At the webpage menu click on ‘Login or Register’ and then ‘Register’.
    2. Enter a valid email address and fill in the required information.
    3. Log in to Galaxy when prompted.
    4. Open the email sent to the email address you provided in the step ‘2’ and click on the link provided to activate your account.

  2. Uploading data to Galaxy (Figure 5)
    1. RNA-Seq analysis using Salmon requires RNA-Seq data (fastq file format) and the transcriptome (fasta file format). The transcriptome for S. carpocapsae can be downloaded from WormBase ParaSite website as follows.
      1. Log on to parasite.wormbase.org/index.html.
      2. Click on ‘Downloads’.
      3. Search for ‘Steinernema carpocapsae’ on the list.
      4. Under ‘Full-length Transcripts’, click on ‘FASTA’ to download the gene sequences of S. carpocapsae in fasta format.
    2. Paired-end S. carpocapsae RNA-seq data from Lu et al. (2017) can be downloaded at GEO under accession GSE89961.


      Figure 5. Galaxy interface to demonstrate sections B-C of Data analysis. User uploads data at ‘Get Data’ as indicated at top left (section B of Data analysis). Salmon is run for gene expression quantification as shown in bottom left (section C of Data analysis). Data can be downloaded from ‘History’ section on right side of page (Step C11 of Data analysis).

    3. Data can be uploaded to Galaxy using two methods:
      1. Locally from your computer:
        1. On Galaxy webpage under ‘Tools’ menu, click on ‘Get Data’ and select ‘Upload File from your computer’.
        2. Click on ‘Choose local file’, and select the transcriptome fasta file. Under the ‘Type (set all):’ drop down menu, select ‘fasta’. Click ‘Start’ to upload the file.
        3. To upload the fastq files, click ‘Choose local file’ again and select the fastq file. Under the ‘Type (set all):’ drop down menu, select ‘fastq’. Click ‘Start’ to upload the file.
      2. From a web-accessible link (preferred method)
        1. On the Galaxy webpage under the ‘Tools’ menu, click on ‘Get Data’ and select ‘Upload File from your computer’.
        2. Click ‘Paste/Fetch data’ and enter the URL of the transcriptome fasta file. Under the ‘Type’ drop down menu, select ‘fasta’. Click ‘Start’ to upload the file.
        3. Click ‘Paste/Fetch data’ and enter the URL of the paired-end fastq files. Be sure that each URL starts on a different line. Under the ‘Type’, drop down menu select ‘fastq’. Click ‘Start’ to upload the files.
        4. When the files are successfully uploaded, the background of the file name field in the history panel on the right of webpage turns green.

  3. Running Salmon (Figure 5)
    Salmon can be used to obtain gene expression levels from RNA-seq data by aligning reads and normalizing the aligned read counts to obtain normalized expression levels in the form of TPM (transcripts per million).
    1. On the ‘Tools’ menu, under ‘NGS: RNA Analysis’, select ‘Salmon’.
    2. Under ‘Select the reference transcriptome’ in the middle panel, select the uploaded transcriptome fasta file.
    3. Under ‘Is this library mate-paired’, select ‘Paired-end’.
    4. For ‘Mate pair 1’, select the uploaded mate pair 1 fastq file.
    5. For ‘Mate pair 2’, select the uploaded mate pair 2 fastq file.
    6. The parameters for Salmon should be set according to the specific type of RNA-seq data to be analyzed which is determined by the library preparation protocol used. An important parameter that needs attention is ‘Specify the strandedness of the reads’. The Lu et al. (2017) RNA-seq data is unstranded, therefore the value for the above parameter should be set to default ‘Not stranded (U)’. Also, under both ‘Perform sequence-specific bias correction’ and ‘Perform fragment GC bias correction’, select ‘Yes’. Select default values for the remaining parameters. For a full description of the parameters please refer to Salmon manual (https://combine-lab.github.io/salmon/).
    7. Click ‘Execute’ to start the analysis.
    8. The status of the job is shown under a new field in the ‘History’ panel to the right.
    9. Click the refresh button to get an updated status of the job. The time for the submitted job to finish varies depending on the queue size on Galaxy and the number of reads uploaded. Usually it takes less than one hour for a Salmon job to finish.
    10. The job field background in the history panel on the right side of the webpage turns green when the job completes successfully.
    11. Once complete, a tab-delimited text file providing the gene/isoform names, a number of reads mapped to the genes/isoforms, and TPM (relative abundances) can be downloaded from the job field in the history panel.
    12. The gene expression results from step 11 can be further analyzed using tools provided through Galaxy such as DESeq2 (Love et al., 2014). e.g., differentially expressed genes/isoforms between experimental conditions/treatments can be identified.

Notes

  1. For embryos, we have also used 0.3% Triton-X for the lysis buffer.
  2. In our hands, 8 out 10 samples pass the first quality check using the BioAnalyzer.
  3. The lysis buffer must be made with sterile Eppendorf tubes, spatulas, and pipette tips.
  4. A new pipette tip should be used for each worm during collection.
  5. Samples that sequence poorly, with less than 1 million reads, or that correlate poorly with other biological replicates are excluded from analysis.

Recipes

  1. Lysis buffer stock (Shaham et al., 2006)
    Notes:
    1. Lysis buffer stock can be stored indefinitely wrapped in foil at room temperature.
    2. Aliquot Triton-X (100%) to a 1.5 ml Eppendorf tube and warm to 35 °C and use as described in Recipe.
    20 µl 1 M Tris-HCl pH 8.0
    20 µl Triton-X (100%)
    200 µl Tween-20 (10%)
    2 µl 0.5 M EDTA
    1.628 ml nuclease free water
    Total 1.871 ml

Acknowledgments

We would like to thank all members of the Mortazavi and Dillman labs for helpful discussions. This work was supported by an NIH New Innovator Award to A.M. (NIGMS DP2 GM111100). The authors declare that they have no conflicts of interest or competing interests.

References

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  8. Macchietto, M., Angdembey, D., Heidarpour, N., Serra, L., Rodriguez, B., El-Ali, N. and Mortazavi, A. (2017). Comparative transcriptomics of Steinernema and Caenorhabditis single embryos reveals orthologous gene expression convergence during late embryogenesis. Genome Biol Evolution 9(10): 2681-2696.
  9. Patro, R., Duggal, G., Love, M. I., Irizarry, R. A. and Kingsford, C. (2017). Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods 14(4): 417-419.
  10. Picelli, S., Bjorklund, A. K., Faridani, O. R., Sagasser, S., Winberg, G. and Sandberg, R. (2013). Smart-seq2 for sensitive full-length transcriptome profiling in single cells. Nature Methods 10(11): 1096-1098.
  11. Picelli, S., Faridani, O. R., Bjorklund, A. K., Winberg, G., Sagasser, S. and Sandberg, R. (2014). Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc 9(1): 171-181.
  12. Shaham, S. (2006). WormBook: Methods in cell biology. In: WormBook (Ed.). The C. elegans Research Community. WormBook.
  13. Trombetta, J. J., Gennert, D., Lu, D., Satija, R., Shalek, A. K. and Regev, A. (2014). Preparation of single-cell RNA-Seq libraries for next generation sequencing. Curr Protoc Mol Biol 107: 4.22.21-17.

简介

大多数线虫是小蠕虫,缺乏足够的RNA用于常规的RNA-seq协议,而没有汇集成千上万的个体。 我们已经调整了Smart-seq2协议来排序单个蠕虫的转录组。 虽然针对Steinernema carpocapsae和Caenorhabditis elegans幼虫以及胚胎开发,但该方案应该适用于其他线虫物种和小无脊椎动物。 另外,我们介绍如何使用Galaxy在线环境分析RNA-seq结果。 我们预计这种方法将有助于研究野生型和突变体背景个体线虫的基因表达差异。

【背景】低输入RNA-seq方案和扩增试剂盒,例如Smart-seq(Takara Bio,USA,Inc)和SuperAmp(Miltenyl Biotec,Inc),已经越来越多地开发和商业化,作为对低输入RNA-基于小组织,单一微生物和单细胞的seq研究。这些研究经常探索并解决特定群体(例如细胞群体,复杂组织或微生物群体)的个体中的异源基因表达。针对微生物(如线虫)的低输入RNA-seq方案的改进和适应将通过允许在单一线虫水平上分析基因表达异质性而极大地有益于线虫领域。在这里,我们已经调整了单细胞RNA-seq方案Smart-seq2(Picelli等人,2013和2014; Trombetta等人,2014),对于单线虫RNA测序。我们成功地在昆虫寄生线虫Steinernema carpocapsae(Lu等人,2017)的转录组学分析中以及在分析个体胚胎和来自两个Steinernema的L1幼虫和两个Caenorhabditis物种,包括C. (Macchietto等人,2017),但是该协议可以适用于任何种类的线虫。虽然这个协议将工作在线虫没有已经测序的基因组或转录组,我们限制我们的计算分析生物与发表的基因组注释,如S。 carpocapsae (Dillman 等,2015)。我们需要单线虫RNA测序作为一种方法来规避低RNA输入样品的工作限制。例如,我们的很多体内实验限制了我们可以利用的线虫数量。单线虫RNA-seq使我们能够有效地从这些线虫获得高分辨率的基因表达数据。该协议也使我们能够收集单个胚胎,以绘制线虫胚胎发育的时间过程,用于跨越多个物种的比较转录组学。低输入RNA-seq方案的发展和进步将有助于调查人员规避与使用单个生物体和专门/有限样品相关的问题。

关键字:RNA-seq, 转录, 秀丽隐杆线虫, 斯氏线虫

材料和试剂

  1. 手套
  2. 8孔,无核酸酶,0.2-ml带帽的薄壁PCR管(SARSTEDT,产品目录号:72.985.002和65.989.002)
  3. 针25 G 1.5英寸常规(BD,PrecisionGlide,目录号:305127)
  4. Qubit TM测定管(Thermo Fisher Scientific,Invitrogen TM,目录号:Q32856)。
  5. 移液器提示
  6. 1.5 ml Eppendorf管
  7. 刮刀
  8. 70%的乙醇或RNA酶
  9. 蛋白酶K(QIAGEN,目录号:19131)
  10. RNasin核糖核酸酶抑制剂(RNase抑制剂)(Promega,目录号:N2611)
  11. UltraPure DNase / RNase free蒸馏水(Thermo Fisher Scientific,Gibco TM,产品目录号:10977015)
  12. Oligo-dT 30 VN引物(从IDT(https://www.idtdna.com/site)订购):5'-AAGCAGTGGTATCAACGCAGAGTACT 30 VN-3' /> 注意:该寡核苷酸与包含poly(A)尾巴的所有RNA退火。该寡核苷酸的3'端含有'VN',其中'N'是任何碱基,'V'是A,C或G.两个末端核苷酸是将寡核苷酸锚定到poly(A)尾巴并避免不必要的长腺苷段的扩增。将寡核苷酸溶解在TE缓冲液中至最终浓度为100μM。将此寡核苷酸在-20°C储存6个月。
  13. dNTP混合物(每种10mM)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0192)
  14. Superscript II逆转录酶试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:18064014)
  15. LNA修改的TSO(从Exiqon( http://www.exiqon.com/ )订购) > 5'-AAGCAGTGGTATCAACGCAGAGTACATrGrG + G-3'
    注意:在5'末端,这个TSO携带一个通用的引物序列,而在3'末端,有两个核糖鸟苷(rG)和一个LNA-修饰的鸟苷(+ G)以促进模板转换。溶于TE缓冲液中的TSO可以在-80℃下以100μM等分试样储存6个月。避免反复的冻融循环。
  16. 甜菜碱(BioUltra≥99.0%)(Sigma-Aldrich,目录号:61962)
  17. 氯化镁(MgCl 2,无水)(Sigma-Aldrich,目录号:M8266)
  18. Kapa HiFi HotStart ReadyMix(Kapa Biosystems,产品目录号:KK2602)
  19. IS PCR oligo(从IDT(https://www.idtdna.com/site)订购)
    5'-AAGCAGTGGTATCAACGCAGAGT-3'
    注:该寡核苷酸在RT后的扩增步骤中充当PCR引物。将寡核苷酸溶解在TE缓冲液中至最终浓度为100μM。这种寡核苷酸可以在-20°C下保存6个月。
  20. Agencourt Ampure XP珠(Beckman Coulter,目录号:A63881)
  21. 乙醇99.5%(体积/体积)(Kemethyl,目录号:SN366915-06)
  22. Qubit TM dsDNA HS测定试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:Q32854)
  23. 安捷伦高灵敏度DNA试剂盒(Agilent Technologies,目录号:5067-4626)
  24. 缓冲液PM(QIAGEN,目录号:19083)
  25. Nextera DNA文库制备试剂盒(24个样品)(Illumina,目录号:FC-121-1030)
  26. QIAquick PCR纯化试剂盒(50)(QIAGEN,目录号:28104)
  27. Phusion高保真PCR主混合物与HF缓冲液,500反应(新英格兰生物实验室,目录号:M0531L)
  28. Tris-HCl pH 8.0(Thermo Fisher Scientific,Invitrogen TM,目录号:AM9850G)
  29. Triton X-100(Sigma-Aldrich,目录号:T9284)
  30. EDTA pH 8.0(Mediatech,目录号:46-034-Cl)
  31. 聚山梨酯20,Acros Organics TM(Tween 20)(Acros Organics,目录号:233362500)
  32. RNaseZap(Thermo Fisher Scientific,Invitrogen TM,产品目录号:AM9780)
  33. 测序指标列表(Buenrostro et al。,2015)(参见补充文件

设备

  1. 移液器
  2. 微型离心机,带8头PCR管的头部
  3. 漩涡
  4. 热循环仪
  5. 立体显微镜
  6. Qubit 荧光计
  7. Agilent 2100生物分析仪(Agilent Technologies,型号:Agilent 2100,目录号:G2938C)
  8. 磁力支架96(Thermo Fisher Scientific,目录号:AM10027)

程序

警告:RNA很容易被RNA酶降解,在冰上不是很稳定!因此,使用70%的乙醇或去离子水清洗所有表面,经常更换手套,并对所有使用的材料进行消毒。
裂解的样品不应该在冰上保存超过一个小时,因为mRNA会降解 注意:


  1. 该协议可用于所有幼虫阶段,dauers和线虫的胚胎。
  2. 每只虫子都应该使用新的无菌针。



  1. 从培养板分离和裂解线虫
    注意:在收集蠕虫的当天准备新的缓冲区。
    1. 用蛋白酶K制备裂解缓冲液(见方案末尾的食谱),将46.8μl裂解缓冲液与3.2μl蛋白酶K混合,总量为50μl。
    2. 从步骤A1将18μl裂解缓冲液与蛋白酶K与2μlRNase抑制剂合并,共20μl裂解缓冲液。
    3. 使用蛋白酶K和RNasin核糖核酸酶抑制剂裂解缓冲液裂解蠕虫。
    4. 建立一个线虫收集站(图1A)。
    5. 用去离子水洗三次线虫:
      1. 要从培养板洗线虫,加入1毫升的水板。

      2. 旋转板三次驱逐线虫。
      3. 用移液管将蠕虫移至1.5 ml Eppendorf管。
      4. 简单地减少。
      5. 去除上清液,用水冲洗1毫升。
      6. 重复步骤A5d和A5e两次或直到水变清。
    6. 使用移液器在2μl水中收集一个蠕虫并转移到PCR管的壁上(图1B)。用25号针头切割蜗杆(图1C-1D)(视频1)。
      注意:切割建议所有幼虫阶段。请注意,我们并没有削减胚胎。

      视频1

    7. 加入2μl裂解缓冲液,含蛋白酶K和RNasin核糖核酸酶抑制剂。
    8. 轻敲管混合,并在微型离心机中旋转离心管。
    9. 在PCR循环仪上使用以下程序在热循环仪上孵育样品。
      1. 65°C-10分钟
      2. 85°C-1分钟
      3. 4°C
    10. 将样品转移到冰上。
    11. 添加1μL寡聚-dT VN引物(10μM)和1μLdNTP混合到每个管。轻弹管混合,并下降管。

    12. 在72°C孵育热循环仪上的样品3分钟
    13. 从热循环仪取样,立即放在冰上。


      图1.准备和切割单个线虫以进行裂解A.用于分离,切割和制备RNA-seq的单个线虫的设置的图片。 B.将移液管尖端分离的单个线虫的图片转移至待切割的PCR管中。 C.在2μlDEPC处理的水中的单个线虫的图片。 D.使用25号针头将PCR管中的单个线虫切成两半的照片。在图B-D中,单独的线虫以红色圈出。

  2. Smart-seq2 mRNA的逆转录(RT)
    1. 准备逆转录主混合物(RT MM)。 (不需要DNase)


    2. 向每个PCR管中加入5.7μlRT MM。
    3. 在微型离心机中迅速涡旋并旋转。
    4. 穿上热循环仪并运行以下程序:

      注:停止点。存放样品-20°C。

  3. Smart-seq2 PCR扩增(图2)
    1. 将样品放在冰上。准备KAPA HiFi主混音。



    2. 加入15μlKAPA HiFi主混合物
    3. 穿上热循环仪并运行以下程序:

      注:停止点。样品可以无限期地储存在-20°C。


      图2.适用于各个线虫的Smart-seq2协议概述。 由Picelli 等人修改(2014)。 1.线虫被切割并裂解以释放总RNA。 2. Oligo(dT)在mRNA的3'末端结合聚(A)尾(程序的A部分)。 3. cDNA的第一链由MMLV逆转录酶(RT)合成,其在cDNA的3'端添加非模板引导的胞嘧啶。这些胞嘧啶用于锚定LNA修饰的TSO。然后逆转录酶使用TSO作为模板来完成第一链(程序的B部分)。 4. ISPCR引物与第一链的3'末端退火,允许DNA聚合酶结合并合成cDNA的第二链,随后扩增cDNA(程序的C部分)。 5.纯化双链(ds)cDNA并检查质量(方法的D和E部分)。 6.通过将ds cDNA片段化并连接衔接子序列(步骤F部分)将ds cDNA转座子标记。 7.加标记的双链cDNA被清除,8.连接到样本特定的指标(程序G部分)。

  4. cDNA珠清理
    1. 从-20℃取出样品,让它们在室温下静置10分钟。
    2. 从冰箱中取出Ampure XP珠子,高速涡旋1分钟。解决方案应该是同质的。
    3. 将26μlAmpure XP珠粒等分至1.5ml Eppendorf管中。需要将珠加热至室温大约8分钟。
    4. 将样品(约26μl)添加到Ampure XP珠(1:1)中。吸取10次,直到彻底混合。
    5. 用Ampure XP珠在室温下在管架上孵育样品8分钟。
    6. 制成600μl新鲜的80%无核酸乙醇。
    7. 孵育后,将样品置于磁珠架上5分钟。
      注意:珠子将被绑定到感兴趣的cDNA。
    8. 使用移液管移除上清液而不干扰珠子。
      注意:将上清液放入旧样品PCR管中,并保存,直至检测到cDNA浓度。
    9. 将试管放在磁珠架上,向珠中加入200μl80%乙醇。等待30秒。
    10. 用附着在真空管或移液管上的针去除上清液,注意不要弄乱珠子。
      注意:不要让珠子干燥。
    11. 重复步骤D9-D10两次。
    12. 让磁珠在磁力架上干燥5分钟,打开盖子。监视干燥。
    13. 当珠子开始裂开时,从磁铁上取下管子,立即加入17.5微升EB缓冲液到珠子上。吸移10次,彻底混合。该解决方案应该是同质和棕色的。
    14. 将样品在室温下在管架上孵育2分钟。
    15. 将样品放回磁珠架上2分钟,直到液体变清。在磁力架上收集管中的15μl上清液。小心不要打扰珠子。将上清放入干净的带有标签的Eppendorf管中并置于冰上。
      注意:收集的液体中不应该有棕色的规格。如果您吸取任何珠子,将样品放回珠子管并重复步骤D14-D15。
    16. 丢弃磁力架中的管道。

  5. 测量cDNA浓度和样品质量检查
    注意:根据制造商的指导,校准Qubit荧光计。
    1. 用于一个样品的Qubit试剂:
      198微升dsDNA HS缓冲液
      1微升dsDNA HS试剂200x
      1μl的cDNA
      将dsDNA HS试剂,dsDNA HS缓冲液和cDNA加入到Qubit试管中。在微型离心机中迅速涡旋并旋转。

    2. 在黑暗中孵育管2分钟
    3. 将cDNA样品放入Qubit荧光计。在Qubit屏幕上,选择“DNA”,选择“高灵敏度”,然后选择“读取样本”。选择“读取库存浓度”并将体积更改为“1μl”。记录cDNA浓度(ng /μl)。
    4. 按照安捷伦高灵敏度DNA试剂盒的操作说明,在cDNA上运行BioAnalyzer(BA)。
      样品应使用安捷伦高灵敏度DNA试剂盒在BioAnalyzer机器上运行,以生成每个样品中cDNA长度(代表mRNA转录物长度)的分布曲线。如果样品没有降解,您将在500-10,000 bp范围内的片段大小中看到一个清晰的峰,而在较小的片段大小处没有峰,表明由全长mRNA转录产生的全长cDNA(图3A)。如果mRNA转录物降解(可能由于RNA酶污染或RNA处理不当),曲线将显示没有峰或许多小峰(图3B)。


      图3. Smart-seq2 BioAnalyzer(BA)图谱的例子A.理想的BA cDNA文件,显示全长转录本,大约1,000bp大的峰,没有PCR引物污染(70bp)。 B.由高度降解的RNA制备的cDNA的BA图谱导致大量的小片段显示出35至300bp的多个峰。

  6. cDNA的标签(图2)
    注意:
    1. 所有试剂和样品应保存在冰上。
    2. 该协议使用标准的Nextera DNA文库制备试剂盒,而不是Nextera XT DNA样品制备试剂盒。
    3. 使用缓冲液PM是因为它含有醋酸,因此可以处理样品的pH值。

    1. 设置一个加热块到55°C,并加热30μLEB缓冲液。
    2. 计算如何从8μl样品中获得20ng的cDNA(见下面的例子)。


    3. 在PCR管中放置20ng Ampure纯化的PCR产物。加入EB使体积达到8μl。
    4. 在新的PCR管中加入10μl来自Nextera DNA文库制备试剂盒的Tagment DNA缓冲液。

    5. 加Nextera DNA Library制备试剂盒中的2.2μlTagment DNA酶1
    6. 设置一个新的P20移液器至15μL,并通过上下移液10次混合转座酶和转座酶缓冲液。
    7. 将所有混合的转座酶和缓冲液转移到您的cDNA中,然后立即上下移液至少10次,混合均匀。
    8. 将样品置于PCR模块上55℃5分钟以启动标记反应。
    9. 当样本在PCR模块上时,获得QIAquick DNA清除柱,缓冲液PM和PE。加标签的cDNA将通过该柱以从DNA片段中去除酶。
    10. 添加60μL缓冲PM到20μL标记的cDNA。通过上下移液直到溶液变清。
    11. 吸取整个量到QIAGEN旋转柱。
    12. 将QIAGEN柱在12,470×g的离心机中旋转1分钟。 cDNA将粘附在柱中的白色过滤器上,液体将通过收集管。
    13. 将流通物倒入垃圾桶。将色谱柱放回收集管。
    14. 加入750μL的缓冲液PE的列。
    15. 将QIAGEN柱在12,470×g的离心机中旋转1分钟。
    16. 将流通物倒入垃圾桶。将色谱柱放回收集管中。
    17. 将QIAGEN柱在12,470×g的离心机中旋转1分钟。
    18. 将色谱柱放入新的,清洁标有样品名称的Eppendorf管。
    19. 加入30微升温热的EB缓冲液到柱内白色过滤器的中心,注意不要用移液器吸头接触过滤器,或将缓冲液吸到柱壁上。
    20. 让列坐1分钟。
    21. 在12,470×g的离心机中旋转离心柱1分钟。
    22. 取下色谱柱并保持流通。
    23. 流通是标记的cDNA。
      注意:在这一步,样品可以交给设备进行测序。如果研究人员添加条形码,请按照以下步骤操作。

  7. 接头连接片段的扩增(图2)
    1. 在PCR管中加入以下试剂:


    2. 将样品放到PCR循环仪上并运行以下程序(Buenrostro et。,2015):

      注:停止点后,样品可以无限期地储存在-20°C。

  8. DNA库珠子清理
    按照过程D中所述的步骤进行以下更改:
    1. 步骤D3,使用70μlAmpure XP珠(1:1)。
    2. 步骤D4,加样(〜70μl)。
    3. 步骤D14,加入30μlEB。
    4. 步骤D16,收集27.5μl上清液。

  9. DNA文库浓度的测量和样品质量检查
    1. 按照程序E中的步骤使用Qubit获取DNA文库浓度。
    2. 运行BioAnalyzer来检查图书馆的质量。 (可选,但强烈推荐!)
      样品应在BioAnalyzer上运行以检查程序F中标签的质量。完全标记的文库将具有在200bp附近具有清晰峰的BioAnalyzer谱(图4A)。部分加标签的文库将具有150至1,000bp的均一平台的BioAnalyzer谱,并且可能具有小于150bp或大于1,000bp的一些较小的峰(图4B)。

      图4.标记和文库制备后的Smart-seq2 BioAnalyzer配置文件示例:一种。已经被完全标记的样品的一个例子显示了大约200bp的一个峰。 B.没有完全标注的样本的例子显示了从150-1,000bp的广泛分布的片段。

  10. 库测序
    图书馆可以交给测序机构。样品使用双末端43个碱基对读数进行测序。双端测序提高了数据集的质量,使阅读更准确地与参考基因组进行比对。也可以使用单端读取对库进行测序。每个样本应测序至少1000万次,以可靠地检测每一百万个转录本(TPM)。

数据分析

分析Smart-seq2数据
通常将RNA-seq文库测序至10-20万个读段的深度以可靠地描绘目的样品中的基因表达水平。尽管STAR(Dobin等人,2013),Kallisto(Bray等人,2016)和Salmon(Patro等人,2013)等快速可靠的软件。,2017)已经被开发用于分析读取和获得基因/同种型表达水平,高通量数据的分析通常需要计算和数据存储资源超出典型台式或膝上型机器的能力。这些任务需要访问高性能计算集群或诸如Galaxy的在线资源(Afgan等人,2016)。 Galaxy通过用户友好的在线界面提供免费的分析工具和计算/存储资源。对使用任何不同的测序方法产生的数据进行测序可以上传到具有合适分析工具的Galaxy,以获得可解释的结果。在这里,我们提供了一个详细的教程,上载Steinernema carpocapsae RNASeq数据到银河和获得基因表达水平使用三文鱼。

  1. 创建一个用户帐户
    Galaxy资源的使用需要用户帐户。 Galaxy可以注册一个免费的用户账号。使用任何网页浏览器登录到 usegalaxy.org
    1. 在网页菜单点击“登录或注册”,然后“注册”。
    2. 输入一个有效的电子邮件地址并填写所需的信息。
    3. 根据提示登录到Galaxy。
    4. 打开发送到您在步骤“2”中提供的电子邮件地址的电子邮件,然后单击提供的链接以激活您的帐户。

  2. 上传数据到Galaxy(图5)
    1. 使用三文鱼的RNA-Seq分析需要RNA-Seq数据(fastq文件格式)和转录组(fasta文件格式)。 S的转录组。可以从WormBase ParaSite网站下载carpocapsae ,如下所示。
      1. 登录到 parasite.wormbase.org/index.html
      2. 点击“下载”。
      3. 在列表中搜索'
      4. 在“全长转录本”下,点击“FASTA”下载 S的基因序列。 carpaocapsae 以fasta格式。
    2. 配对结束的 S。 carpocapsae 来自Lu et al。(2017)的RNA-seq数据可以在GEO下载 GSE89961
    3. 数据可以通过两种方式上传到Galaxy:
      1. 本地从您的计算机:
        1. 在“工具”菜单下的Galaxy网页上,点击“获取数据”并选择“从计算机上传文件”。
        2. 点击“选择本地文件”,然后选择transcriptome fasta文件。在“Type(全部设置):”下拉菜单中,选择“fasta”。点击“开始”上传文件。
        3. 要上传fastq文件,请再次单击“选择本地文件”并选择fastq文件。在“Type(全部设置)”下拉菜单中,选择“fastq”。点击“开始”上传文件。
      2. 从网络访问链接(首选方法)
        1. 在“工具”菜单下的Galaxy网页上,点击“获取数据”并选择“从计算机上传文件”。
        2. 点击“粘贴/取数据”并输入转录组fasta文件的URL。在“类型”下拉菜单中,选择“fasta”。点击“开始”上传文件。
        3. 点击“粘贴/抓取数据”并输入配对结束的fastq文件的URL。确保每个网址都在不同的行上。在“类型”下,下拉菜单中选择“fastq”。点击“开始”上传文件。
        4. 当文件成功上传时,网页右侧历史记录栏中文件名字段的背景变成绿色。


          图5. Galaxy界面演示数据分析的B-C部分。用户在“获取数据”上传数据,如左上方(数据分析的B部分)所示。运行鲑鱼进行基因表达定量,如左下方(数据分析的C部分)所示。数据可以从页面右侧的“历史”部分下载(数据分析的步骤C11)。

  3. 运行三文鱼(图5)
    鲑鱼可以通过比对读数并标准化比对读数来获得RNA-seq数据的基因表达水平,以获得TPM形式的标准化表达水平(每百万转录本)。
    1. 在“工具”菜单上的“NGS:RNA分析”下,选择“三文鱼”。
    2. 在中间面板的“选择参考转录组”下,选择上传的转录组fasta文件。
    3. 在“这是图书馆配对”,选择“配对结束”。
    4. 对于“配对1”,选择上传的配对1 fastq文件。
    5. 对于“配对2”,选择上传的配对2 fastq文件。
    6. 鲑鱼的参数应根据待分析的RNA-seq数据的具体类型设置,这是由所使用的文库制备方案确定的。需要注意的一个重要参数是“指定读取的关联性”。 Lu等人。 (2017)RNA-seq数据是非保留的,因此上述参数的值应设置为默认的“不搁置(U)”。另外,在“执行序列特异性偏好校正”和“执行片段GC偏差校正”下,选择“是”。选择其余参数的默认值。有关参数的完整说明,请参阅鲑鱼手册( https://combine-lab.github.io / salmon / )。
    7. 点击“执行”开始分析。
    8. 工作状态显示在右侧“历史记录”面板的一个新字段中。
    9. 点击刷新按钮以获得作业的更新状态。提交的作业完成时间取决于Galaxy上的队列大小和上载的读取次数。通常鲑鱼工作完成不到一个小时。

    10. 。当作业成功完成后,网页右侧的历史记录面板中的作业领域背景变成绿色
    11. 一旦完成,提供基因/同种型名称的标签分隔文本文件,映射到基因/同种型的多个读段和TPM(相对丰度)可以从历史记录栏中的工作领域下载。
    12. 步骤11的基因表达结果可以使用通过Galaxy提供的工具进一步分析,例如DESeq2(Love等人,2014)。例如,可以鉴定实验条件/处理之间差异表达的基因/同种型。

笔记

  1. 对于胚胎,我们也使用了0.3%的Triton-X作为裂解缓冲液。
  2. 在我们的手中,8个样品中的8个通过使用BioAnalyzer的第一次质量检查。
  3. 裂解缓冲液必须使用无菌Eppendorf管,刮刀和移液枪头。

  4. 每个蠕虫在采集时都应该使用新的枪头
  5. 排序不佳,读数少于1百万的样本或与其他生物学重复数据不相关的样本不包含在分析中。

食谱

  1. 裂解缓冲液(Shaham et al。,2006)
    注意:
    1. 裂解缓冲液储液可以在室温下无限期地包裹在箔中。
    2. 将Triton-X(100%)分装到1.5ml Eppendorf管中并温热至35℃并按照配方中所述使用。
    20μl1 M Tris-HCl pH 8.0
    20μlTriton-X(100%)
    200μlTween-20(10%)
    2μl0.5 M EDTA
    1.628毫升无核酸酶水
    总共1.871毫升

致谢

我们要感谢Mortazavi和Dillman实验室的所有成员进行有益的讨论。这项工作得到了美国国立卫生研究院新的创新者奖的支持。 (NIGMS DP2 GM111100)。作者声明他们没有利益冲突或利益冲突。

参考

  1. 阿富汗,E.,贝克,D,van den Beek,M.,Blankenberg,D.,Bouvier,D.,Cech,M.,Chilton,J.,Clements,D.,Coraor,N.,Eberhard,C Gruning,B.,Guerler,A.,Hillman-Jackson,J.,Von Kuster,G.,Rasche,E.,Soranzo,N.,Turaga,N.,Taylor,J.,Nekrutenko, Goecks,J。(2016)。 银河平台提供可访问,可复制和协作的生物医学分析:2016年更新。 >核酸研究44(W1):W3-W10。
  2. Bray,N.L.,Pimentel,H.,Melsted,P。和Pachter,L。(2016)。 近乎最优的概率RNA-seq定量。 34(5):525-527。
  3. Buenrostro,J.D。,Wu,B.,Chang,H.Y。和Greenleaf,W.J。(2015)。 ATAC-seq:全基因组染色质可达性检测方法 Curr Protoc Mol Biol 109:21 29 21-29。
  4. Dillman,AR,Macchietto,M.,Porter,CF,Rogers,A.,Williams,B.,Antoshechkin,I.,Lee,MM,Goodwin,Z.,Lu,X.,Lewis,EE,Goodrich-Blair, H.,Stock,SP,Adams,BJ,Sternberg,PW和Mortazavi,A。(2015)。 Steinernema的比较基因组学揭示了深度保守的基因调控网络。 Genome Biol 16:200。
  5. Dobin,A.,Davis,C. A.,Schlesinger,F.,Drenkow,J.,Zaleski,C.,Jha,S.,Batut,P.,Chaisson,M.和Gingeras,T. R.(2013)。 STAR:超快速通用RNA-seq定位仪 生物信息学 29(1):15-21。
  6. Love M.I.,Huber W.,Anders S.使用DESeq2(2014)对RNA-seq数据的倍数变化和扩散的中度估计。
    基因组生物学 15(12):550
  7. Lu,D.,Macchietto,M.,Chang,D.,Barros,M.M.,Baldwin,J.,Mortazavi,A。和Dillman,A.R。(2017)。 活性昆虫病原线虫感染性幼虫释放致命毒液蛋白 PLoS Pathog < 13(4):e1006302。
  8. Macchietto,M.,Angdembey,D.,Heidarpour,N.,Serra,L.,Rodriguez,B.,El-Ali,N.和Mortazavi,A。(2017)。 比较的转录组学> Steinernema 和 Caenorhabditis 单胚胎显示在晚胚胎发生期间直向同源基因表达收敛。 Genome Biol Evolution 9(10):2681-2696。
  9. Patro,R.,Duggal,G.,Love,M. I.,Irizarry,R.A。和Kingsford,C。(2017)。 三文鱼提供了对转录物表达的快速和偏见感知量化。 Nat Methods 14(4):417-419。
  10. Picelli,S.,Bjorklund,A.K。,Faridani,O.R。,Sagasser,S.,Winberg,G。和Sandberg,R。(2013)。 Smart-seq2用于在单个细胞中进行敏感性全长转录组分析 Nature Methods 10(11):1096-1098。
  11. Picelli,S.,Faridani,O. R.,Bjorklund,A. K.,Winberg,G.,Sagasser,S.和Sandberg,R.(2014)。 使用Smart-seq2从单个细胞获取全长RNA-seq Nat Protoc 9(1):171-181。
  12. Shaham,S。(2006)。 WormBook:细胞生物学的方法在:WormBook(Ed。)。 C.线虫研究社区。 WormBook 。
  13. Trombetta,J. J.,Gennert,D.,Lu,D.,Satija,R.,Shalek,A.K。和Regev,A。(2014)。 制备用于下一代测序的单细胞RNA-Seq文库。 Curr Protoc Mol Biol 107:4.22.21-17。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Serra, L., Chang, D., Macchietto, M., Williams, K., Murad, R., Lu, D., Dillman, A. R. and Mortazavi, A. (2018). Adapting the Smart-seq2 Protocol for Robust Single Worm RNA-seq. Bio-protocol 8(4): e2729. DOI: 10.21769/BioProtoc.2729.
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