GSM3466615: O5_ISC: RNAseq_OLD_ISC_3; Mus musculus; RNA-Seq
Villus cell suspensions for RNA-seq were stained in 2% fetal calf serum/PBS with 1:200 APC-conjugated Epcam antibodies (CD326, eBioscience 17-5791-82), 1:1000 AF488-conjugated CD45 antibodies (Biolegend 103122) and 1:1000 AF488-conjugated CD31 antibodies (Biolegend 102414) for 15 minutes at room temperature, washed 3 times and resuspended in 2% FCS/PBS with 3.2µg/ml ROCK-inhibitor (Y-27632, Sigma). Epcamhigh CD31- CD45- DAPI- cells were sorted on BD Aria III SORP cell sorter with 100 µM nozzle. ISC (GFPhigh DAPI-) for RNA-seq were isolated from crypt suspensions of Lgr5-GFP+ mice and processed as above except the staining step. 60-100 thousand cells were sorted into RLT buffer for subsequent RNA-isolation according to manufacturer's protocol with the Qiagen RNeasy Micro kit with on-column DNA digestion. Quantity and quality were measured with an Agilent Bioanalyzer Eukaryote Total RNA Pico assay. Libraries were generated from 35 ng total RNA according to NEB Ultra II Directional RNA Library Preparation Kit for Illumina (E7760), Poly(A) mRNA magnetic isolation module (E7490) and multiplex oligos (E7335) manuals with the following modifications: 14 PCR-amplification cycles were performed. SPRI select beads were substituted with Seramag Speedbeads (Thermo scientific 65152105050250) for size selection steps.
GEO Accession
GSM3466615