RNA-seq was performed using Smart-Seq2 (29) with minor modifications. Cells were sorted into 19 μl of TCL buffer and 1 μl proteinase K (20 mg/ml; Qiagen), and their RNA was purified by a 2.2× SPRI cleanup with RNAClean XP magnetic beads (Agencourt) before reverse transcription. For the microfluidic implementation of the protocol, Tween 20 (Teknova) was added to all reactions at a final concentration of 0.5%. For mRNA capture, a biotinylated oligo (/5BiosG/ - AAGCAGTGGTATCAACGCAGAGTAC-30T-VN) (Integrated DNA Technologies) was attached to streptavidin magnetic beads (New England Biolabs) following the manufacturer’s protocol. The beads were then used to capture mRNA from the lysates and were washed with 10 mM tris-HCl (pH 7.5), 0.15 M LiCl, 1 mM EDTA, and 0.5% Tween 20. The beads were then resuspended in the reverse transcription mix, following the same steps as the published protocol. cDNA processed on the benchtop and microfluidic device were amplified for 18 and 22 cycles, respectively. After amplification and cleanup, libraries were quantified using a Qubit fluorometer (Invitrogen), and their size distributions were determined using the Agilent Bioanalyzer 2100. After normalizing the amplicon concentrations to 0.1 to 0.2 ng/ml, sequencing libraries were constructed using the Nextera XT DNA Library Prep Kit (Illumina), following the manufacturer’s protocol. All RNA-seq libraries were sequenced with 38 × 37 paired-end reads using a MiniSeq or NextSeq (Illumina).

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.