GSM9001162: IEC - Transgenic - DSS - Replicate 6; Mus musculus; RNA-Seq
For colon tissue RNA extraction, 5mm distal colon was placed in a 2 ml microcentrifuge tube containing one stainless steel bead (5mm mean diameter), add 150 μl Buffer RLT per tube and operate the TissueLyser II for 5 min at 20 Hz to generate tissue homogenate. Keep on ice for 10mins, centrifuge at 12000 g for 10 min at 4 °C, collect supernatant. For IECs, directly lysis with 150 μl Buffer RLT. Next, total RNA was isolated by using the RNeasy Micro Kit (74004, Qiagen) according to the manufacturer's instructions. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.