alias: GSM4412514
title: GSM4412514: P2_rep2; Mus musculus; RNA-Seq
library_construction_protocol: Small intestinal crypts of EF1a-OSCAR mice were isolated and a single cell suspension derived by an 20min incubation with TrypLE at room temperature. Cells were stained with anti-EpCAM PE-Cy7 for 15mins on ice before washing and staining with Sytox Blue dead cell stain. FACS sorting was performed on a BD FACS ARIA III. 10.000 cells of each population were sorted and RNA extracted using TRIzol Reagent. Integrity and concentration of isolated RNA was determined using Qubit 4 Fluorometer (Thermo Fisher) and Fragment Analyzer (Agilent). Poly-A library preparation was performed using TruSeq RNA Library Prep Kit V2 (Illumina) according to manual. Briefly, poly-A containg mRNA was enriched using magnetic beads, first strand synthesis of cDNA was done using SuperScript III Reverse Transcriptase (Thermo Fisher), second strand synthesis was performed using the second strand master mix (TruSeq RNA Library Prep Kit V2 (Illumina)) and the DNA was cleaned up using Agencourt AMPure XP Beads (Beckman Coulter). After end repair and dA tailing, adaptors were ligated and the library was further enriched using the PCR Master Mix (TruSeq RNA Library Prep Kit V2 (Illumina)). Quality and concentration of the library was determined using Qubit 4 Fluorometer (Thermo Fisher) and Fragment Analyzer (Agilent). cDNA libraries were pooled and 20pM loaded into the NextSeq 500 (Illumina) Sequencer using the NextSeq 500/550 High Output Kit v2.5 (75 cycles).
identifiers.namespace: GEO
identifiers.id: GSM4412514