title: GSM1937896: Bsubtilis_230_RNAseq; Bacillus subtilis; OTHER
library_construction_protocol: Cell culture was rapidly filtered in 250 mL increments at 37C 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). For nascent RNA purification, the clarified lysate was added to 0.5 mL anti-FLAG M2 affinity gel (Sigma Aldrich). The affinity gel was washed twice with lysis buffer supplemented with 1 mM EDTA before incubation with the clarified lysate at 4 °C for 2.5 h with nutation. The immunoprecipitation was washed 4 × 10 mL with lysis buffer supplemented with 300 mM KCl, and bound RNAP was eluted twice with lysis buffer supplemented with 1 mM EDTA and 2 mg/mL 3XFLAG peptide (Sigma Aldrich). Nascent RNA was purified from the eluate using the miRNeasy kit (Qiagen). The full library construction protocol was described in detail previously (Larson et al., Science 2014). Purified nascent RNA was ligated to a 5' adenylated DNA oligo and fragmented to generate RNA ranging from 30-100 nt. The RNA was reverse transcribed, and the single-stranded DNA circularized and PCR amplified. For RNA-seq, the total RNA was fragmented and dephosphorylated with T4 PNK before continuing onto the reverse transcription, circularization, and PCR steps.
identifiers.namespace: GEO
identifiers.id: GSM1937896