Illumina NovaSeq 6000 paired end sequencing; RNA-Seq from the colon tissues of DSS treated mice receiving either EP4-D, a selective E-type prostanoid receptor 4 agonist or vehicle
RNA-Seq from the colon tissues of DSS treated mice receiving either EP4-D, a selective E-type prostanoid receptor 4 agonist or vehicle
Colon tissues were harvested from euthanized mice. The rectal tissues were discarded and 0.2 to 0.3 cm of the distal colon was collected in RNA later solution and immediately snap frozen in liquid nitrogen. 10 week old mice were challenged with 3% DSS in drinking water. Mice were divided into two groups, one received sterile PBS as control and the other received 1mg.kg-1 of EP4-D dissolved in sterile PBS once daily via oral gavage. The volume of gavage was set such that desired dosage was achieved by gavaging (body weight in grams X 10) in microliters. For e.g a 20g mouse received an oral bolus of 200 microliters* (either vehicle or compound)  Total tissue RNA was extracted using the column based RNA extraction kit, Total RNA Kit, peqGOLD (VWR GmbH, Munich, Germany) as per manufacturer's instructions. From the RNA sample to the final data, each step, including sample test, library preparation, and sequencing, influences the quality of the data, and data quality directly impacts the analysis results. To guarantee the reliability of the data, quality control (QC) is performed at each step of the procedure. After the QC procedures, mRNA from eukaryotic organisms is enriched using oligo(dT) beads. For prokaryotic samples, rRNA is removed using a specialized kit that leaves the mRNA. The mRNA from either eukaryotic or prokaryotic sources is then fragmented randomly in fragmentation buffer, followed by cDNA synthesis using random hexamers and reverse transcriptase. After first-strand synthesis, a custom second-strand synthesis buffer (Illumina) is added with dNTPs, RNase H and Escherichia coli polymerase I to generate the second strand by nick-translation. The final cDNA library is ready after a round of purification, terminal repair, A-tailing, ligation of sequencing adapters, size selection and PCR enrichment. Library concentration was first quantified using a Qubit 2.0 fluorometer (Life Technologies). Insert size was checked on an Agilent 2100 and quantified using quantitative PCR (Q-PCR).