title: GSM7030363: ecol_hs_abx_03_v17 [mixed sample]; Escherichia coli; OTHER
library_construction_protocol: Microbial cell pellets were resuspended in equal volume of LET (25 mM Tris pH 8.0, 100 mM LiCl, 20 mM EDTA) and water saturated Phenol pH 6.6 (Thermo Fisher),  lysed with acid washed glass beads (Sigma-Aldrich) and vortexing for three minutes in MultiMixer. Lysates were supplemented  with equal volumes of phenol/chloroform isoamyl alcohol pH 4.5 (25:24:1) and nuclease free water, l two-minute vortexing followed by centrifugation. Resulting aqueous phase was purified in two steps using phenol-chloroform isoamyl alcohol (25:24:1) followed by chloroform. After centrifugation, the clean aqueous phase was precipitated with sodium acetate-ethanol. Standard and random fragmented 5PSeq libraries were generated as described in PMID: 26046441 with minor modifications using 150-9000ng total RNA as an input. To prepare random fragmented samples (negative controls), ribosomal RNA was depleted from DNA-free RNA and subsequent fragmented by incubating five minutes at 80 ̊C in fragmentation buffer (40mM Tris Acetate pH 8.1, 100mM KOAc and 30mM MgOAc). Reaction was purified using 2 volumes of RNACleanXP beads (Beckman Coulter) as recommended by the manufacturer. Free 5'OH sites were re-phosphorylated using 5 Units of T4 Polynucleotide kinase (PNK, NEB) and incubated at 37 ̊C for 60 minutes as recommended by the manufacturer. Re-phosphorylated fragmented RNA was purified using Phenol:Chloroform: Isoamyl Alcohol (24:25:1), followed by sodium acetate-ethanol precipitation. From this step forward, procedures for random fragmented and standard 5PSeq library preparation merge. RNA was Ligated to either rP5_RND or rP5_RNA oligo (specified in characteristics protocol).    containing unique molecular identifiers). Ribosomal RNA was depleted using Ribozero rRNA removal kit (Illumina) suitable for Bacteria and Yeast. rRNA depleted sample was purified using 1.8V of Ampure beads (Abcam), fragmented using heat (80˚C) for 5 min in 5x Fragmentation Buffer (200mM Tris Acetate pH 8.1,500mM KOAc,150mM MgOAc) and reverse transcribed using random hexamers to prime. Resulting cDNA was bound to streptavidin beads (M-280), subjected to enzymatic reactions of DNA end repair, fill-in of adenine to 5' protruding ends of DNA fragments using Klenow Fragment (NEB). Common adaptor (P7-MPX) was ligated and 5PSeq Libraries were amplified by PCR (15- 17 cycles). Purified samples were quantified using Qubit (Thermo Fisher), library size was estimated from bioanalyzer traces. 5PSeq Libraries were pooled by mixing equal amounts of each sample, following enrichment of fragments of the length between 300-500nt.   HT-5PSeq Libraries were generated as recently described (bioRxiv 2020.06.22.165134; doi: https://doi.org/10.1101/2020.06.22.165134). In brief, DNA-free RNA was ligated with RNA oligos containing unique molecular identifiers. Ligated RNA was reverse transcribed priming with oligos containing a random hexamer and an Illumina compatible region. RNA was eliminated by addition of NaOH. Ribosomal RNA was depleted by adding in-house rRNA DNA oligo depletion mixes (table 2) to the cDNA and performing a duplex-specific nuclease (DSN, Evrogen) treatment. rRNA depleted cDNA was PCR amplified (15-17 cycles). Depletion of ribosomal RNA with Ribozero Illumina (for bacteria and yeast) was done after the single-stranded RNA ligation step. Ribosomal depleted RNA was purified and reverse transcribed using the same oligos as stated above, and then amplified by PCR. Libraries were quantified by fluorescence (Qubit, Thermo Fisher), size estimated using an Agilent Bioanalyzer and sequence using a NextSeq500 Illumina sequencer.