title: GSM5415986: efae_poxTA_LZD_rep01; Enterococcus faecalis; OTHER
library_construction_protocol: RNA was extracted via phenol-chlorofrom extraction, briefly; 300 ul LET-phenol was added to cell pellets and vortexed with an equal volume of 0.1 mm glass beads to lyse cells. 250 ul of water and 250 ul of acidic phenol/chlorophorm was added before votexing  for a further 2 minutes. RNA was then extracted from the aqueous phase with an acidic phenol/chloraform and chloroform extraction step and ethanol precipitated in the presence of NaOAc pH 5.2. RNA was assessed by running a 2 % agarose gel and quantified by Qubit. HT-5PSeq libraries were prepared as described in (Zhang and Pelechano, Cell Reports Methods, Volume 1, Issue 1,2021). Briefly, 4µg total RNA, total RNA from Enterococcus faecalis was used and ligated over night at 16˚C to r5P_RNA_ oligo (CrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrU rNrNrNrNrNrNrNrN) and subsequent purified using 1.8x volumes of RNAClean XP beads (Beckman Coulter). Ligated RNA was then reverse transcribed using random hexamer  (5Pseq-RT, GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNN, 20µM) to prime. Left over RNA was degraded using NaOH. Ribosomal RNA was removed using previously described rRNA DNA oligo depletion mixes (Huch, Nersisyan et al doi: https://doi.org/10.1101/2021.04.08.439066), following duplex-specific nuclease (DSN, Evrogen) degradation. rRNA depleted cDNA was amplified by PCR (17 cycles) and enriched for fragments with the range of 300-500nt using Ampure XP. Size selected HT-5P Libraries were quantified by fluorescence (Qubit, Thermo Fisher), size estimated using an Agilent Bioanalyzer and sequenced using a NextSeq2000 Illumina sequencer (100cycles P3 kit).