GSM6261007: KPCS398_Nx; Mus musculus; RNA-Seq
Organoids were harvested and RNA was extracted using the NucleoSpin® RNA kit (Macherey-Nagel) according to the manufacturer's instructions. For each organoid line, 2-3 wells of organoids from a 24 well plate were harvested in lysis buffer RA1 (RNA extraction kit, MN) supplemented with TCEP (Tris(2-carboxyethyl)phosphine hydrochloride). For library preparation, the quantity and quality of the isolated RNA was determined with a Qubit® (1.0) Fluorometer (Life Technologies, California, USA) and a Bioanalyzer 2100 (Agilent, Waldbronn, Germany). The TruSeq Stranded mRNA Sample Prep Kit (Illumina, Inc, California, USA) was used in the succeeding steps. Briefly, total RNA samples (1 μg) were ribosome depleted and then reverse-transcribed into double-stranded cDNA with Actinomycin added during first-strand synthesis. The cDNA samples were fragmented, end-repaired and polyadenylated before ligation of TruSeq adapters. The adapters contain the index for multiplexing. Fragments containing TruSeq adapters on both ends were selectively enriched with PCR. The quality and quantity of the enriched libraries were validated using Qubit® (1.0) Fluorometer and the Bioanalyzer 2100 (Agilent, Waldbronn, Germany). The product is a smear with an average fragment size of approximately 360 bp. The libraries were normalized to 10nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20. Cluster generation and sequencing involved application of the TruSeq SR Cluster Kit v4-cBot-HS or TruSeq PE Cluster Kit v4-cBot-HS (Illumina, Inc, California, USA) using 8 pM of pooled normalized libraries on the cBOT.