title: GSM8793312: proximal colon3_WT; Mus musculus; RNA-Seq
library_name: GSM8793312
library_construction_protocol: 1 cm of each indicated segment of the intestine from WT and GsdmdΔIEC mice was isolated. Total RNA was isolated and purified using TRIzol reagent (Invitrogen) following the manufacturer's procedure. The amount and purity of each RNA sample was quantified with NanoDrop ND-1000 (NanoDrop) and RNA integrity was assessed by a Bioanalyzer 2100 (Agilent) with RNA Integrity Number >7.0, and further confirmed by electrophoresis with denaturing agarose gel. Poly (A) RNA was purified from 1 μg total RNA using Dynabeads Oligo (dT) (25-61005, Thermo Fisher) using two rounds of purification, followed by fragmentation using the Magnesium RNA Fragmentation Module (cat.e6150, NEB) at 94℃ for 5-7 min. Cleaved RNA fragments were reverse-transcribed to create cDNA with SuperScript™ II Reverse Transcriptase (cat.1896649, Invitrogen), then were next used to synthesize U-labeled second-stranded DNAs with E. coli DNA polymerase I (cat.m0209, NEB), RNase H (cat.m0297, NEB), and dUTP solution (cat.R0133, Thermo Fisher). After PCR amplification, the average insert size for the final cDNA library was 300 ± 50 bp.