GSM1191856: Rib 3; Bacteroides cellulosilyticus; RNA-Seq
RNAprotect Bacteria Reagent (Qiagen) was used to stabilize both frozen fecal pellets (experiment NM601) and in vitro cultures (experiment NM602; added prior to pelleting cells) per the manufacturer's instructions. Samples were then mechanically disrupted using acid-washed glass beads in a Mini-BeadBeater-8 in the presence of acid phenol:chloroform:IAA (125:24:1), after which ethanol precipitation was performed. RNA was then further purified using a MEGAclear kit, tested for integrity by gel electrophoresis, and shown to be lacking any contaminating genomic DNA by PCR. rRNA depletion was subsequently performed via two rounds of processing with the MICROBExpress kit and two rounds of rRNA capture using custom oligos attached to magnetic beads (Dynabeads). First-strand cDNA synthesis was then performed using random hexamer priming with SuperScript II, after which second-strand synthesis was achieved using E. coli DNA polymerase in the presence of RNase H. Libraries were prepared according to a slightly modified version of the protocol accompanying the Illumina Genomic DNA Sample Prep Kit. Briefly, cDNA was sonicated in a BioRuptor XL water bath sonicator, cleaned up and concentrated using a Qiagen PCR Purification column, and end-repaired using Klenow DNA polymerase. The blunt DNA was treated with Klenow fragment (exo minus) to add an adenine overhang, and the A-tailed molecules were ligated to the relevant Illumina adapter sequence. Non-barcoded adapters were used to construct libraries from experiment NM601 samples. Adapters incorporating an 8 bp barcode were used to construct libraries from experiment NM602 samples. Adaptered-DNA was then size-selected by agarose gel electrophoresis. Fragments of the appropriate size were PCR amplified and purified, after which the purified PCR products were loaded on an Illumina flow cell for cluster generation. Libraries were sequenced on either the Illumina Genome Analyzer IIx (experiment NM601) or the Illumina HiSeq 2000 (experiment NM602) using the manufacturer's protocols.
GEO Accession
GSM1191856