Illumina NovaSeq 6000 paired end sequencing; Temporal transcriptomic changes during DSS colitis development and resolution.
Temporal transcriptomic changes during DSS colitis development and resolution.
Mice were euthanized and colon tissues were harvested. The rectal tissues were discarded and 0.2 to 0.3 cm of the distal colon tissue was excised and snap frozen in liquid nitrogen. Mice recieved 3% DSS in drinking water and were monitored for disease development using disease activity scores. This is a combined score for weight loss, stool consistency and occult blood scores. Occassionally mice also recieved minicolonoscopic examination to confirm colitis. Total RNA from the tissues were harvested using the column purification method using the Total RNA Kit, peqGOLD (VWR GmbH, Munich, Germnay) as per the manufacturer's instructions. From RNA samples extraction to final data obtained, every step of procedures containing samples testing, library construction and sequencing would directly affect the quality and quantity of data, which determines the bioinfomatic analysis results. Novogene promises to strictly control on all production data with high quality output and ensure the raw data accurate and reliable. Briefly, mRNA from Eukaryote organisms is purified from total RNA using poly-T oligo-attached magnetic beads (For prokaryotes, mRNA was purified through the removal of rRNA by using kit). The mRNA is first fragmented randomly by addition of fragmentation buffer. Then first strand cDNA is synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis is subsequently performed using DNA Polymerase I and RNase H. Double-stranded cDNA is purified using AMPure XP beads. Remaining overhangs of the purified double-stranded cDNA are converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure is ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments are purified with AMPure XP system (Beckma Coulter, Beverly, USA). Finally, the final library is gotten by PCR amplification and purification of PCR products by AMPure XP beads.