GSM4474396: Nfkb2 knockout treated 2; Mus musculus; RNA-Seq
Colons were surgically removed from the indicated mice subsequent to euthanization, longitudinally cut open, washed with PBS, and then cut into 2-5 mm small pieces. These pieces were rocked on a shaker platform submerging in HBSS containing 30mM EDTA that led to the detachment of IECs. IECs were collected by centrifugation and subjected to biochemical analyses. The purity of isolated IECs, determined as CD45.2-EpCAM+ cells, was found to be typically ~ 90% in flow cytometric analyses. Total RNA from mouse colonic epithelial cells was isolated following the double extraction protocol:  RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction (TRIzol, Thermo Fisher Scientific) followed by a Qiagen RNeasy Micro clean-up procedure according to the manufacturer's protocol. All RNAs were analyzed using an Agilent Bioanalyser (Agilent, Santa Clara, CA, USA) for quality assessment with RNA Integrity Number (RIN) ranging from 7.2-9.2 and a median RIN of 8.8. cDNA libraries were prepared from 2 ng total RNA starting material and 1 ml of a 1:50,000 dilution of ERCC RNA Spike in Controls (Ambion® Thermo Fisher Scientific) using the SMARTSeq v2 protocol (Picelli et al., 2014) with the following modifications: 1. Use of 20 mM TSO; 2. Use of 250 pg cDNA with 1/5 reaction of Illumina Nextera XT kit (Illumina, San Diego, CA, USA). The length distribution of the cDNA libraries was monitored using a DNA High Sensitivity Reagent Kit on the Perkin Elmer Labchip (Perkin Elmer, Waltham, MA, USA).