title: GSM8494938: b_subtilis_methyl_rep2; Bacillus subtilis; OTHER
library_construction_protocol: Cells were collected and pelleted at 3,000 xg for 10 minutes, resuspended in 250 μL lysis buffer (30 mM Tris pH 7.0, 10 mM EDTA, 10 mg/mL lysozyme), and incubated at 25 ºC for 30 minutes. Total RNA was then extracted (1 mL of TRIzol; Invitrogen) and purified with on-column DNase treatment (Monarch Total RNA Miniprep; NEB). Click labeling and RNA enrichment. Total RNA was chemically fragmented at 94 ºC for 2 minutes to yield an average size of ~200 nucleotides (Magnesium RNA Fragmentation Module; NEB). RNA fragments were purified (silica-based SPE, using Monarch RNA Cleanup Kit; NEB).  The cross-linked RNA fragments were then labeled with magnetic azide click beads via Cu(I)-catalyzed azide–alkyne cycloaddition. The cycloaddition was generally performed with 0.5 µM of heat-denatured RNA, 0.5 µM medium magnefy azide click beads (Bang Laboritories, Inc.), 240 µM Copper(II)-TBTA complex (Lumiprobe), 500 µM fresh ascorbic acid (Sigma), and 50% v/v DMSO, in a final reaction volume of 50 µL. All reactions were sparged with nitrogen, incubated at 40 °C for 30 minutes, and finally quenched with 1 µL of 0.5 M EDTA. The cross-link RNA was enriched by resuspending the RNA four times with wash buffer (1M NaCl, 10 mM Tris pH 7.5, 1 mM EDTA, 0.01% TWEEN-20) and a final time in nuclease-free water, the supernatant from each wash was removed via pipette after immobilizing the bead-conjugated RNA using a magnetic separation rack (NEB). MaP reverse transcription. RNA was subjected to mutational profiling reverse transcription with random nonamer primers, with the addition of robust denaturation step for rRNA analysis . Bead-conjugated RNA was mixed with 100 ng of random nonamer primer (NEB), and 20 nmol of dNTPs (NEB) and then denatured at 90 ˚C for 5 minutes followed by incubation at 4 ˚C for 2 minutes. MaP-RT buffer (6 mM MnCl2, 1 M betaine, 50 mM Tris (pH 8.0 at 25 ˚C), 75 mM KCl, and 10 mM fresh DTT) was added to the RNA solution and incubated at 25 ˚C for 2 minutes. SuperScript II Reverse Transcriptase (1 μL, 200 units; Invitrogen) was added and the reverse transcription reaction was performed according to the following temperature program: 25 °C for 10 min, 42 °C for 90 min, 10 × [50 °C for 2 min, 42 °C for 2 min], 72 °C for 10 min (Smola et al., 2015). The resulting cDNA was purified via magnetic pull-down using a magnetic separation rack (NEB). Library preparation and sequencing. Double-stranded DNA was prepared from the reverse transcription reaction product (NEBNext Ultra II Non-directional RNA Second Strand Synthesis Module; NEB) and DNA sequencing libraries were prepared using the manufacturer protocol (NEBNext Ultra II DNA Library Prep with Sample Purification; NEB). Libraries were then quantified (Qubit high-sensitivity dsDNA assay; Invitrogen and High sensitivity D1000 screentape; Agilent) and pooled as an equimolar sequencing library. The library was sequenced using 2x110 paired-end sequencing on an Illumina NextSeq 1000 instrument (P2 200 cycle sbs reagents; Illumina).