title: GSM3076171: Calf 35 P5 Organoids; Bos taurus; RNA-Seq
library_construction_protocol: To extract RNA from the organoid cultures, organoids were treated with 1.0 ml Corning Cell Recovery Solution (Corning) and incubated for 1 h at 4ºC with constant shaking at 120 rpm.   The suspension was then centrifuged at 1,200 x g for 5 min, and the pellet washed twice in 1.0 ml ice-cold PBS.  The pellet was then lysed in 600 µl of the RLT buffer (Qiagen, Germany) containing 10 µg/ml 2-mercaptoethanol (Sigma-Aldrich, UK), and homogenized using a Qiashredder homogenizer (Qiagen).  Total RNA was then extracted using a RNeasy mini kit (Qiagen) according to the manufacturer's instructions which included a DNase digestion step to remove genomic DNA.  The RNA was eluted into RNase-free water. 3 µg of RNA (10 wells) was used for RNA seq. 7 libraries were prepared using the TruSeq stranded total RNA-seq library preparation kit (Illumina) with one round of RiboZero Gold treatment The libraries were pooled and the pool was sequenced on 3 lanes on a HiSeq 4000 instrument with 150-base paired end reads to a depth of 147-191M read pairs per sample.
attributes.tag: GEO Accession
attributes.value: GSM3076171