Illumina HiSeq 2000 paired end sequencing; Transcriptomic analysis of the distal colon of wild type C57BL/6N mice and mice harboring a mutation (S839I) in Gucy2c
Transcriptomic analysis of the distal colon of wild type C57BL/6N mice and mice harboring a mutation (S839I) in Gucy2c
A 1cm piece from the end of the colon was left and 1 cm towards the proximal end was harvested and placed in 1 ml of Trizol and snap frozen.  Samples were then processed using the Qiagen RNeasy mini kit as per manufacturer's instructions. Total RNA was isolated and sent for RNAseq analysis RNA was quantified by QUBIT 3 Fluorometer using Ribogreen Dye. 1 microgram of total RNA was used to enrich the mRNA Oligo dT magnetic beads, and the mRNA was chemically fragmented in a magnesium-based buffer at 94°C for 15 minutes. The fragmented mRNAs were primed with random hexamers, and reverse transcribed to form cDNA. The cDNA fragments obtained were cleaned up by using AM pure beads. The DNA underwent end repair wherein the mix converts the overhangs resulting from fragmentation into blunt ends. The 3' to 5' exonuclease activity of end repair mix removes the 3' overhangs and polymerase activity fills in the 5' overhangs. To the blunt ended fragments adenylation was performed by adding single 'A' nucleotide to the 3' ends. To the adenylated fragments loop adapters were ligated and cleaved with uracil-specific excision reagent (USER) enzyme. The samples were further purified using AMPure beads and The DNA was then enriched by PCR with 12 cycles using NEBNext Ultra II Q5 master mix, illumina universal primer and sample-specific octamer primers. The amplified products were cleaned up by using AM pure beads and the final mRNA library was eluted in 15 microlitres  of 0.1X TE buffer.
Experimental Factor: genotype
Gucy2c S839