GSM1367310: Ecoli_WT_RNAseq; Escherichia coli; RNA-Seq
Cell culture was rapidly filtered in 250 mL increments at 37 °C over 0.22 μm nitrocellulose ﬁlters (GE) and frozen in liquid nitrogen to simultaneously halt all transcriptional progress. Frozen cells (100 μg) were pulverized on a Qiagen TissueLyser II mixer mill 6 times at 15 Hz for 3 min in the presence of 500 μL frozen lysis buffer (20 mM Tris pH 8, 0.4% Triton X-100, 0.1% NP-40, 100 mM NH4Cl, 50 U/mL SUPERase•In (Ambion)) and 1X protease inhibitor cocktail (Complete, EDTA-free, Roche), supplemented with 10 mM MnCl2. The lysate was resuspended on ice by pipetting. RQ1 DNase I (110 U total, Promega) was added and incubated for 20 min on ice. The reaction was quenched with EDTA (25 mM final), which releases polysomes from the transcript and reduces contamination from ribosomal RNA and ribosome-associated tRNAs. The lysate was clarified at 4 °C by centrifugation at 20,000 g for 10 min. The lysate was loaded onto a PD MiniTrap G-25 column (GE Healthcare) and eluted with lysis buffer supplemented with 1 mM EDTA. Total RNA was purified from the clarified lysate using the miRNeasy kit (Qiagen). The full library construction protocol was described in detail previously (Churchman et al., Nature 2011). Purified total RNA was fragmented and dephosphorylated with T4 PNK, and then ligated to a 5' adenylated DNA oligo to generate RNA ranging from 30-100 nt. The RNA was reverse transcribed, and the single-stranded DNA circularized and PCR amplified.
GEO Accession
GSM1367310