GSM7233771: ∆SI3* biol rep3 OD0.49; Escherichia coli; OTHER
Frozen cells were pulverized on a Retsch MM400 Mixer Mill (canisters pre-cooled in liquid nitrogen) 6 times at 15 Hz for 3 min in the presence of 400 µL frozen lysis buffer (20 mM Tris-HCl, pH=8.0, 0.4% Triton-X 100, 0.1% NP-40, 100 mM NH4Cl, 10 mM MnCl2, 250 Unit RNaseIn (Promega), 1X protease inhibitor cocktail, 0.4 mg/mL puromycin). Spike-in cells were thawed on ice and added proportional to the E. coli cell amount (500 µL*OD E.coli) to homogenize with E. coli cells. The lysate was resuspended on ice with extra 5 mL of lysis buffer by gentle pipetting. RQ1 DNase I (110 Unit total, Promega) was added and incubated for 15 min on ice. The reaction was quenched with EDTA (25 mM final), which releases ribosomes from the transcript. The lysate was clarified at 4 °C by centrifugation at 25,000 xg for 10 min and incubated with 0.5 mL anti-FLAG M2 affinity resin (Sigma-Aldrich) for 3 hours at 4 °C. The resin was washed with 10 mL Wash Buffer (20 mM Tris-HCl, pH=8.0, 0.4% Triton-X 100, 0.1% NP-40, 300 mM KCl, 1 mM EDTA) 4 times at 4 °C and the RNAP was eluted with 600 µL Elution Buffer (20 mM Tris-HCl, pH=8.0, 0.4% Triton-X 100, 0.1% NP-40, 2 mg/mL FLAG peptide (Sigma-Aldrich)). Nascent RNA was extracted from the eluate with miRNeasy Mini Kit (Qiagen) following manufacturer's instructions. The nascent RNA sample was further purified by EtOH precipitation, dissolved in DEPC-treated water and store at –80 °C. The adaptor oligo(/5Phos/CTGTAGGCACCATCAAT/3ddC/, 6 µM) was 5' adenylated by incubating with 80 µM ATP, 6 µM Mth RNA ligase (NEB), 25% (v/v) PEG 8000 in 1X NEB Buffer 1 at 65 °C for 4 hours, then 85 °C for 5 min. Nascent RNA (16.7 ng/µL, normally 1.5 µg for each sample) was ligated to the adaptor by incubating with 1X T4 RNA ligase buffer (NEB), 25% PEG 8000, 6.7 Unit/µL T4 RNA ligase 2, truncated (NEB), 2 µM adaptor, 1.3 Unit/µL RNaseIn at 37 °C for 3 hours. The ligation was stopped by adding 16 mM EDTA. The adaptor-ligated RNA was alkaline fragmented by incubating with 6 mM Na2CO3, 44 mM NaHCO3, 1 mM EDTA at 95 °C for 35 min (time may vary depending on the freshness of the alkaline solution) and the RNA was precipitated by isopropanol precipitation with NaOAc (pH=5.5) and GlycoBlue coprecipitant (Thermo Fisher). The fragmented, adaptor-ligated RNA was run on a 15% TBE urea polyacrylamide gel (Novex, Thermo Fisher) and RNA species ranging from 50 to 100 nt was excised and gel extracted. The RNA was reversely transcribed into cDNA by mixing with 1X First Strand Buffer (Thermo Fisher), 0.5 mM dNTPs, 0.3 µM RT adaptor (/5Phos/AGATCGGAAGAGCACACGTCTGAAC/iSp18/CACTCA/iSp18/CCTACACGACGCTCTTCCGATCTTCCGACGATCATTGATGGTGCCTACAG), 0.6 Unit/µL SuperaseIn, 5 mM DTT, 10 Unit/µL Superscript III (Thermo Fisher) and incubating at 50 °C for 60 min. The RNA template in the cDNA was eliminated by adding 0.1 M NaOH and incubating at 95 °C for 20 min followed by adding 0.1 M HCl. The cDNA was run on a 10% TBE urea polyacrylamide gel (Novex, Thermo Fisher) and DNA species ranging from 100 to 200 nt was excised and gel extracted. The cDNA library was circularized by incubating with 1X Reaction Buffer (Lucigen), 50 µM ATP, 2.5 µM MnCl2, 5 Unit/µL CircLigase (Lucigen) at 60 °C for 1 hour followed by 80 °C for 10 min. The cDNA libraries were amplified with Q5 DNA polymerase (NEB) PCR system using dual indexed primers for NovaSeq 6000 (Illumina) sequencing platform using 150-nt paired-end sequencing.