RNA library preparation

Total RNA extraction from CiBER-seq yeast pellets                                                   

(modified from: Isolation of Total RNA from Yeast Cell Cultures; Manuel Ares; Cold SpringHarb Protoc; doi: 10.1101/pdb.prot071456)

Materials:

• Sodium Acetate 3M pH 5.5 (Ambion #AM9740)
• EDTA 0.5M pH 8.0(Ambion #AM9260G)
• Nuclease-Free Water
• Nuclease-Free Microcentrifuge Tubes
• Phase-lock tubes (useful but not necessary for this protocol)
• Thermomixer
• PCA pH 6.6 (Ambion #AM9730)
• Chloroform (Fisher Chemical C607SK-1)
• Ethanol, 100% and 70%, diluted in nuclease-free H2O
• GlycoBlue (Ambion #AM9515 )

Prep Work:

Place thermomixer in hood at 65°C.

Chill microcentrifuge to 4°C. (all centrifugations should take place at 4°C) Make AE buffer

Thaw Yeast Pellets:

1. Thaw frozen yeast pellets on ice from a turbidostat run. The pellets should contain ~250-500 million yeast cells, corresponding to 25-50mL of spun down haploid yeast turbidostat culture of OD = 1)

Extraction:

1. Resuspend the pelleted cells in 400μL of AE buffer and transfer to a 1.5-mL RNase free microcentrifuge tube.
2. Add 40μL of 10 % SDS and 400μL of PCA. Place lid lock on tube. Vortex the mixture quickly and then place the tube into a thermomixer set at 65°C.
3. Incubate the cells for 15 min at 65°C on the thermomixer at 2000rpm.
4. Remove from thermomixer, and incubate the cells for 5 min on ice with the lid lock still on.
5. Centrifuge the sample at 14,000 rpm (or top speed) at 4°C in a microcentrifuge for 5min. After centrifugation, the organic phase will separate from the aqueous phase. The RNA will be in the upper aqueous phase.
6. Remove the aqueous phase, making sure not to pipette any of the white material in between the phases (its better to get less aqueous phase, then to accidentally get some of the interphase) and add this to a separate RNAse-free tube. Discard the lower organic phase
7. Add 400μL of chloroform to this top (aqueous) phase. Mix by gently shaking, but do not vortex. This step removes water- dissolved organic solvents from the aqueous phase. Centrifuge again at 14,000 rpm at 4°C for 5 min.
8. Add another 400 μL of chloroform to the tube and gently shake again. Centrifuge one last time at 14,000 rpm at 4°C for 5 min.
9. Carefully transfer the top aqueous phase into a new 1.5mL microcentrifuge tube.

Precipitation:

1. Add 50 μL of 3 M NaOAc, pH 5.2, and 1uL of Glycoblue to the aqueous phase.
2. Add 100% ethanol up to 1.5 mL. Do not overfill the tube. Make sure that the cap fits and then mix well by inverting several times. The RNA should precipitate readily at this concentration, so it is not necessary to chill the ethanol. (If needed, this is a good stopping point, RNA is fairly stable in ethanol at -20°C)
3. Centrifuge at 14,000 rpm at 4°C for 5–10 min. You should see an RNA pellet at the bottom of the tube.
4. Pour off/pipette off the ethanol and rinse the pellet with 70% ethanol. Centrifuge at 14,000rpm at 4°C for 5min.
5. Pour off/pipette away the ethanol and allow the pellet to air-dry. (Roughly 20-ish mins) (Place the tubes sideways in a rack to prevent debris from falling in, make sure all the residual ethanol is gone from the pellet. You can use a quick centrifuge spin and P2 to pipet away residual ethanol to speed up the evaporation.

Water Resuspension:

1. Resuspend the pellet in 100μL of Nuclease-free H2O by vortexing. It may take some time for the RNA to come back into solution, so be patient and keep the RNA on ice. Carefully inspect the sample visually to make sure that there are no clear chunks of RNA precipitate still floating and that the side of the tube to which the RNA was pelleted is clean. Do not proceed until you are reasonably convinced that you have a solution, and not a suspension, of RNA.

2. Once the pellet is completely in solution, read the A260, A270, A280, and A320in a spectrophotometer or take a spectrum in a nanospectrophotometer. If the RNA is pure, the A260:A280 ratio should be 1.8–2.2, the A320 (particulates) should be very low, and the A270 should never be higher than the A260 value; otherwise, there is phenol contamination. Expect a yield of 100uL at roughly 5 ug/uL.

3. Store the RNA in H2O at –80°C. It should be stable for several years.

Dual Promoter CiBER-seq library prep                                                   

Introduction

This protocol is specifically for preparing RNA sequencing libraries from yeast transformed with dual barcoded gRNA libraries, such as: https://benchling.com/s/seq-S5cQupYmmAS5YGu3hWZU or similar dual citrine/mCherry expression cassettes. This plasmid is constructed with 5nt identifiers for both divergent expression cassettes to allow pooling of multiple yeast strains and/or libraries within a single experiment.

Materials

› Yeast Pellets, stored at -80°C or flash frozen

› Hot Phenol Extraction reagents

› See accompanying Total RNA extraction protocol

› Protoscript II reverse transcriptase Kit (NEB #M0368L)

› NEB-next dual indexing primers (NEB #E7600S)

› Primer: RM511: GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTtGGTCCAGTCTTGTTACCAGACAACC

› Primer: RM810: ACACTCTTTCCCTACACGACGCTCTTCCGATCT

› Primer: RM812: GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTcCGGCGCCTACAACGTCAACATC

› RNAse A (Thermo Scientific #EN0531)

› RNAse H (NEB #M0297S)

› Turbo DNAseI (optional) (Invitrogen #AM2238)

› Zymo RNA clean and concentrate kit (optional) (Zymo #R1017)

› Zymo DNA clean and concentrate kit (Zymo #D4013)

› Q5 polymerase (NEB #M0491L)

› Thermocycler

› Tape Station/Bioanalyzer + necessary Tape reagents

› AMPure XP beads or similar DNA size selection (if necessary, based on results of library prep)

Procedure

Library prep on expressed barcodes

1. Thaw 25-50 mL of frozen pelleted yeast from the turbidostat:
Its good to harvest, pellet, and freeze down (at -80°C) extra yeast so that you have something to go back to in case something in the process fails. For reference, the yeast in the turbidostat should be OD roughly 0.8-1.2 Given that 1mL of OD600 = 10^7 cells, 25 mL (~250 million cells) should be enough to cover a 240,000 barcoded guide library with about 1000x coverage.
 

2. Refer to the "Total RNA extraction from CiBER-seq yeast pellets" to purify total RNA. As an optional but recommended step, you may treat with Turbo DnaseI according to manufacturer instructions and RNA column clean to get rid of contaminating DNA.

3. Reverse transcribe 4ug of extracted RNA for each sample using protoscript II manufacturer's protocol. (This will be a 4x scaled reverse transcription reaction, since the protocol recommends 1ug of RNA per 20uL reaction) Use oligo dT to perform the RT reaction.

4. Add 0.5uL RNAse A and 0.5uL RNAse H directly to the RT product and incubate for 1 hour at 37°C. (This is important to get rid of RNA that wasn't reverse transcribed, like rRNA and tRNA, since these can compete with column binding) Column clean the reverse-transcribed DNA with the Zymo DNA clean and concentrate kit according to kit instructions using ssDNA 7:1 ratio binding buffer. Elute in nuclease-free water.

5. Use Q5 polymerase according to manufacturer's instructions to PCR amplify a stretch of ~300nt on the cDNA template containing the expressed barcode. This step adds the i7 annealing site for downstream library prep. Use 6 cycles of extension and 1/2 the purified reverse transcription product as template. Save the other 1/2 column- cleaned reverse transcription product to go back to if necessary. Use primers RM511: GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTtGGTCCAGTCTTGTTACCAGACAACC and RM810: ACACTCTTTCCCTACACGACGCTCTTCCGATCT for the citrine expression cassette (Tm = 70°C) , or RM812: GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTcCGGCGCCTACAACGTCAACATC and RM810:ACACTCTTTCCCTACACGACGCTCTTCCGATCT for the mCherry expression cassette (Tm = 68°C). Use 10 second extension times.

6. Column clean the PCR product according to kit instructions and elute in nuclease-free water.

7. Each round 1 PCR product now has the annealing sites compatible for use with NEB i5/i7 dual index primers. Assign primer pairs to each sample, making sure to choose a set of dual index primers that are compatible for pooled sequencing and will allow the samples to be separated by adapter sequence. Refer to the dual index primer manual for more information. Perform Q5 PCR amplification with 1/2 the column-cleaned product as template and NEB i5/i7 dual index primers according to manufacturer's instructions. Save the other 1/2 column-cleaned product to go back to if necessary. Use annealing temp 72°C, and 10sec extension. Aim for low number of cycles, to avoid overamplification of PCR product. For highly-expressed genes, like those driven by P(PGK1), 6-10 cycles is sufficient, but lower-expressed transcripts may need more cycles.

8. Column clean the PCR product according to kit instructions and elute in nuclease-free water.

9. Run your samples on a Tape Station/Bioanalyzer to determine the concentration and sizing of each amplified barcode library. One sharp peak at the expected amplicon size, ~330bp, indicates a successfully-prepared sample. An additional peak that runs at a high molecular weight may indicate overamplification, as amplicons that melt and re-anneal form bubbles at the mismatched barcode sequence which tend to run slower on the Tape Station. I aim for this over-amplification peak to be <10% the molarity of the ~330bp peak. Over-amplified samples can be re- prepared from the remaining 1/2 round 1 column-cleaned product using fewer PCR cycles. Free primers and primer dimers in the sample should be removed using AMPure XP size selection or similar size selection strategy as they affect the quality of downstream sequencing.

10. Successfully-prepared samples can be pooled and submitted for HiSeq or NovaSeq

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