GSM8465234: rnjA_depletion_rppH_5end_seq; Bacillus subtilis; RNA-Seq
Prior to RNA extraction, cell pellets were washed with 10-15 mL ice cold 10 mM Tris pH 7.0 to remove contaminating nucleic acids that precipitate from 2xYT in methanol. For all 5′ end sequencing samples, RNA was extracted using the RNAsnap protocol and Turbo DNase treated as described for the Rend-seq and 3′ end sequencing experiments. rRNA was depleted from 10 µg of purified RNA with the MICROBExpress Bacterial mRNA Enrichment Kit, per manufacturer's instructions, precipitated, and resuspended in 10 mM Tris pH 7.0. 600 ng rRNA depleted RNA was then ligated to 260 pmol of an RNA adapter for 18 hours at 22 °C using T4 RNA Ligase 1. Following ligation, RNA was fragmented for 2 minutes using RNA fragmentation reagents (ThermoFisher) and fragments of either size 41-71 or 60-90 were isolated through denaturing polyacrylamide gel extraction. RNA fragments were dephosphorylated, polyadenylated, and reverse transcribed following the BaM-seq multiplexed RNA sequencing protocol (Johnson et al., 2023). To add Illumina adapters, pooled cDNA was amplified between 5-10 cycles with primers oDP128 and oDP161 (Johnson et al., 2023) using Q5 DNA polymerase and purified by polyacrylamide gel electrophoresis.