GSM2338942: lactose treatment, replicate B; Bifidobacterium longum; RNA-Seq
Briefly, RNA was obtained using the Ambion RNAqeous kit (LifeTechnologies), with a pretreatment using 250 μl lysozyme (50 mg/ml final; Sigma) and 125 μl mutanolysin (1000 units/ml; Sigma), at 37˚C for 10 min. Total RNA was immediately subjected to DNase treatment with the Turbo DNase free (Ambion) using 3 μl of DNase I for 1 hour at 37º C. RNA integrity was checked in a Bioanalyzer using an Agilent RNA Nano Chip, with a minimal RIN (RNA Integrity Number) of 7. Genomic DNA contamination was evaluated by qPCR and universal bacterial primers Uni334F and Uni514R53. Ribosomal RNA depletion was achieved using the Ribo-Zero magnetic kit Bacteria (Epicentre, Madison WA). Manufacturer instructions were followed (using 28 or 26 μl of RNA depending on the concentration). mRNA was purified with the Qiagen RNeasy Minelute Cleanup kit, following the instructions presented in the Ribo-Zero protocol for this part. RNA was eluted in 13 µl. One μl of the RNA was checked for removal of 16S and 23S rRNA peaks using the Qubit HS RNA Assay (Agilent). Chromatograms were manually inspected. Messenger RNA was finally converted to cDNA. For first strand cDNA synthesis, the Superscript II Reverse Transcriptase was used (Invitrogen) and second cDNA strand was synthesized using the NEBNext mRNA Second Strand Synthesis Module (New England Biolabs, Ipswich, MA). Then, the MinElute Reaction Cleanup Kit (Qiagen, Valencia CA) was used for cleaning up the DNA. cDNA was quantified using the Qubit High Sensitivity DNA kit (Life Technologies), and fragmented in a Bioruptor (Diagenode). An average fragment length of 300-500 bp was desired for next steps. Sequencing libraries were prepared using the BiooScientific NEXTflex Chip Seq Kit (Bioo Scientific, Austin TX). This kit is Illumina compatible. Ten ng of fragmented cDNA were used. In the manufacturer protocol, option 3 was followed entirely, with the goal to select fragments between 300 and 400 bp in a gel-free protocol. Size selection and clean-up steps were achieved using the Agencourt Ampure XP beads (Beckman Coulter, Brea CA), and a Magnetic Stand-96 (LifeTechnologies). All temperature-sensitive reactions were carried out in a preheated thermocycler. Adapters used (NEXTflex™ ChIP-Seq Barcodes, Bioo Scientific) contained 6-bp indexes for multiplexing. At the last step, ligation products were amplified by PCR with the supplied reagents for 17 cycles. Pooled libraries were sequenced on an Illumina HiSeq 2500. Sequencing was run for 50 cycles, with read length of 50 bp (single reads).
GEO Accession
GSM2338942