title: GSM3069905: PBS7_S2; Clostridioides difficile 630; RNA-Seq
library_construction_protocol: Cells were harvested by centrifugation at 9,000 x g for 5 min at 4°C. Total RNA was extracted from aliquots of ~4.0 E8 cells using the Qiagen RNAprotect® Bacteria Reagent Handbook Protocol 4: "Enzymatic Lysis and Proteinase K Digestion of Bacteria" with modifications.  A mechanical lysis step was incorporated – cells in TE buffer with proteinase K and lysozyme were added to a Lysing Matrix A tube (MP Biomedicals) and treated in a Fastprep FP120 machine (MP Biomedicals) at speed 5.5 for 30s.  The Qiagen protocol was then followed and both on-column and in-solution DNAse digestions, with a final on-column cleanup were used to purify the RNA. RNA samples were confirmed free of contaminating genomic DNA by PCR with gyrA, rpsJ, and adk primers and stored at -70°C until required. For all six RNA samples (3 Faecal water, 3 BHIS control), an RNA integrity number of >9.6 was obtained, with A260/280 values of > 2.0, and 23S:16S rRNA ratios of ≥ 1.4. A 1µg amount of Total RNA was used for rRNA depletion using Ribo-Zero rRNA Removal Kit (Gram-Positive Bacteria) (Illumina, MRZGP126). Illumina stranded whole transcriptome sequencing libraries were prepared using NEBNext Ultra Directional RNA library prep kit for Illumina (NEB, E7420S).  The standard protocol for use with Ribosome Depleted RNA was followed except, after second strand systhesis the samples were precitiated with 1ul (20ng/ µL) glycogen and 1/10th vol. 3M sodium acetate; pellets were washed once with 80% ethanol, followed by 70% ethanol and after air-drying, the pellet was resuspended in 58 µL of water.  The standard protocol for use with Ribosome Depleted RNA was resumed for the remaining steps, except libraries were size selected using Agencourt AMPure XP beads at a 1.5x ratio to retain the smaller sized fraction (~150bp).  NEB Next Multiplex Oligos for Illumina kit (Primer set 1) (NEB, E7335S) was used to generate barcoded multiplex libraries.  Library QC was performed using bioanalyser HS kit (Agilent biotechnologies, 5067-4626).  Libraries were quantified using qPCR (Kapa Biosystems, KK4824). Libraries were pooled at desired concentrations, denatured and loaded for sequencing according to the manufacturer's instructions.  Sequencing was performed over 3 runs on the Illumina MiSeq sequencing platform to generate 2 x 75bp reads.
attributes.tag: GEO Accession
attributes.value: GSM3069905