GSM9500297: sRNA MEF (15A3) Zswim8 knockout clone 2; Mus musculus; miRNA-Seq
Cultured cell lines were scraped from culture dishes into TRI Reagent and RNA was purified according to manufacturer's instructions. Small-RNA sequencing libraries were prepared from 5 μg total RNA. 0.5 fmoles of miR-427-5p (X. tropicalis) and 0.5 fmoles of lsy-6-3p (C. elegans) were added to each sample as spike-ins. Small RNA species were isolated by excising the gel fragment migrating between 18-nt and 32nt radiolabeled internal standards on a 15% polyacrylamide urea gel. Size selected RNA was eluted from the gel, precipitated in ethanol, and ligated to a preadenylated 3¢ adapter (AppNNNNCTGTCTCTTATACACATCTCCGAGCddC) using T4 RNA Ligase 2 KQ mutant (NEB) in a reaction supplemented with 10% polyethylene glycol (PEG 8000, NEB). The 3¢ adapter had 4 random-sequence positions at its 5¢ end to reduce ligation bias. Ligated small RNAs were isolated on a 10% polyacrylamide urea gel, precipitated in ethanol, and ligated to a 5¢ adapter (CCUACACGACGCUCUUCCGAUCUNNNN) using T4 RNA Ligase I (NEB) in a reaction supplemented with 10% PEG. The 5¢ adapter had 4 random-sequence positions at its 3¢ end to reduce ligation bias. Ligated small RNAs were isolated on an 8% polyacrylamide urea gel, precipitated in ethanol, and reverse transcribed with SuperScript III (Invitrogen). Resulting cDNA was amplified using KAPA HiFi DNA polymerase (Kapa Biosystems). Amplified DNA was purified on a 90% formamide, 8% acrylamide gel and submitted for sequencing on the Illumina HiSeq platform. A step-by-step protocol for constructing libraries for small-RNA sequencing is available at http://bartellab.wi.mit.edu/protocols.html.