GSM4038914: CIMEF_CyrKO_8hr_EU; Mus musculus; miRNA-Seq
Cells were rinsed twice with PBS prior to the addition of Trizol. Following addition of the Trizol, cells were scraped off the plate and snap frozen. Total RNA was later extracted as per the manufacturer's protocols. Libraries were generated as detailed at  http://bartellab.wi.mit.edu/protocols.html. Briefly, samples for library preparation were assembled by mixing either 2–5 μg of total RNA (input libraries) or the entire eluate from 5EU pulldown (EU libraries) with size-selection markers and quantitative sequencing standards. Size-selection markers were 18 and 32 nt 5'-end-labeled RNAs. Sequencing standards were added at a level of either 0.1 fmol per 1 μg total RNA (input libraries) or 2.5 amol per sample (EU libraries). Both 3' and 5' ligations were carried out with degenerate adaptors (each containing 4 random nucleotides) in the presence of 10% Polyethylene Glycol and 0.5 μL of Superasin with either T4 RNA-ligase 2, truncated K227Q or T4 RNA-ligase 1, respectively. Reverse transcription was with SuperScript III, and subsequent PCR amplification of the cDNA was with Phusion. Samples were sequenced on an Illumina HiSeq platform with 40 nt single-end reads. RNA-seq samples were prepared using the NEXTflex Rapid Directional mRNA-seq Kit. Briefly, mRNAs were enriched from 10 µg total RNA with NEXTflex Poly(A) Beads. These species were then fragmented, synthesized into cDNA, ligated to adaptors, and PCR amplified as per the manufacturer's protocol. Samples were then sequenced on an Illumina HiSeq platform with 40 nt single-end reads.