title: GSM4314561: WT_24h_rep3; Homo sapiens; RNA-Seq
library_construction_protocol: Qiagen RNEasy For RNA extraction, 8 x 20 µl Matrigel droplets with organoids were harvested and organoid pellets were lysed in RLT buffer. RNA was isolated using the RNeasy Kit (Qiagen) according to the manufacturer's instructions. The quality of the isolated RNA was determined with a Qubit® (1.0) Fluorometer (Life Technologies) and a Bioanalyzer 2100 (Agilent). Only those samples with a 260 nm/280 nm ratio between 1.8–2.1 and a 28S/18S ratio within 1.5–2 were further processed. The TruSeq RNA Sample Prep Kit v2 (Illumina) was used in the succeeding steps. Briefly, total RNA samples (100-1000 ng) were poly-A enriched and then reverse-transcribed into double-stranded cDNA. The cDNA samples were fragmented, end-repaired and polyadenylated before ligation of TruSeq adapters containing 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 Caliper GX LabChip® GX (Caliper Life Sciences). The libraries were normalized to 10nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20. The TruSeq PE Cluster Kit HS4000 or TruSeq SR Cluster Kit HS4000 (Illumina) was used for cluster generation using 10 pM of pooled normalized libraries on the cBOT. Sequencing was performed on the Illumina HiSeq2000 single end 100 bp using the TruSeq SBS Kit HS4000 (Illumina, Inc, California, USA).
attributes.tag: GEO Accession
attributes.value: GSM4314561