GSM7678265: Glc2; Bifidobacterium catenulatum subsp. kashiwanohense; RNA-Seq
Frozen cell pellets obtained from 2 ml of early exponential phase cultures (OD600=0.35) were resuspended in a solution containing 710 µL of the extraction mixture (200 mM NaCl, 20 mM EDTA, 20% SDS), 500 µL of phenol:chloroform:isoamyl alcohol (125:24:1, pH 4.5), and 250 µL of acid-washed glass beads (212-300 µm). Cells were disrupted using Bead Ruptor 12 (Omni International) by alternating 2 min of homogenizing at 6 m/s and 2 min of cooling on ice. Cellular debris was removed by centrifugation (16,000 × g for 10 min), and the aqueous phase was collected. RNA was precipitated with isopropanol and sodium acetate (pH 5.5) at −20 °C overnight, washed twice in ice-cold 70% ethanol, and resuspended in 100 µL of nuclease-free water. Total RNA was subjected to two consecutive DNase treatments (30 min each) with Turbo DNase (Ambion) and Baseline-ZERO DNase (Lucigen), respectively. Each treatment was followed by cleanup using MEGAclear Transcription Clean-Up Kit (Ambion). RNA was quantified via Qubit Broad Range RNA Assay Kit (Invitrogen), and RNA integrity was confirmed by gel electrophoresis in 1 % agarose and 4200 TapeStation System (Agilent). All samples had RIN > 8.0 Ribosomal RNA was depleted with the NEBNext rRNA depletion kit for bacteria (New England Biolabs) and a set of 20 pooled sequence-specific probes for B. kashiwanohense Bg42221_1E1 designed with NEBNext Custom RNA Depletion Design Tool v1.0. Barcoded libraries were made with NEBNext Ultra II directional RNA library prep kit for Illumina (New England Biolabs). Libraries were pooled and sequenced (single-end 75-bp reads) on Illumina NextSeq 500 using the High Output V2 kit (Illumina).