title: GSM3208673: V595_RNASeq; Bos taurus; RNA-Seq
library_construction_protocol: Tissue was minced to a fine slurry in freshly prepared Collagenase solution (0.2% Collagenase A (Roche), 0.05% Trypsin (1:250 powder, 100U/ ml  Gibco/life sciences), Hyaluronidase (Sigma, St Louis MO), 5% Fetal Calf serum, Pen/Strep/Fung (hyclone) or 5µg/ml Gentamycin (Sigma), in DMEM/F12 (Gibco/Life technologies) 10 ng/ml Insulin). Minced tissue was incubated in Collagenase solution (10 ml solution/g tissue) for 3.5 hours at 37C in 50 ml conical tube with slow shaking (120 rpm). Digested tissue was centrifuged for 10 minutes at 453xg at 4°C, supernatant and fat layer discarded, pellet was gently resuspended by pipetting in 5 ml DMEM/F12 without serum. A further 5 ml DMEM/F12 without serum was added and resuspended sample was centrifuged for 10 minutes at 453xg at 4°C, media was discarded and pellet resuspended gently in 10 ml DMEM/F12 and centrifuged for 10 minutes at 453xg at 4°C . Pellet was resuspend gently in 10 ml followed by brief pulls spins at 453xg (centrifuge set to 1 minute @ 453 x g, hit the brakes 2 seconds after 453 xg is reached). Pulls spins were repeated at least 4 time or until very few single cells and mainly epithelial organoid clusters were observed under a microscope. Organoid pellet for RNA-analysis was resuspended in 1 ml TRIzol and stored at -80C.   To Isolate RNA, Macherey-Nagel NucleoSpin miRNA kit for isolation of small and Large RNA was used according to manufacturers protocol (November 2011/rev.02). RNA was analyzed on Bioanlayzer (Agilent) all  RIN were ≥7.5 and stored at -80C till further use. Whole-transcriptome RNA sequencing (RNA-seq) was performed using high quality total RNA (RIN>7) extracted from mammary epithelial organoids to prepare strand-specific, poly-A+ RNA-seq libraries for sequencing on the Illumina platform. To monitor sample and process variability, in addition to sequence metrics, RNA developed by the External RNA Controls Consortium (ERCC) which consists of a mix of 92 polyadenylated RNA transcripts was also added to total RNA at the beginning of the library preparation.  Briefly, poly-A+ mRNA was extracted from 1 μg total RNA using Oligo(dT)25 Dynabeads (Life Technologies, Cat. No. 61002) followed by fragmentation of the mRNA by heat at 94oC for 6 minutes. First strand cDNA was synthesized using the Superscript III reverse transcriptase (Life Technologies, Cat. No. 18080-044) and purified using Agencourt RNAClean XP beads (Beckman Coulter, Cat. No. A63987). During second strand cDNA synthesis, dNTP mix containing dUTP was used to introduce strand-specificity.  For Illumina paired-end library construction, the resultant cDNA was processed through end-repair and A-tailing, ligated with Illumina PE adapters, and then digested with 10 units of Uracil-DNA Glycosylase (NEB, Cat. No. M0280L).  Amplification of the libraries was performed for 13-14 PCR cycles using the Phusion High-Fidelity PCR Master Mix (NEB, Cat. No. M0531L); twelve such 6-bp molecular barcodes were also incorporated during this PCR amplification. The cDNA was purified with Agencourt AMPure XP beads after each enzymatic reaction, and after quantification using the Agilent Bioanalyzer 2100 DNA Chip 7500 (Cat. No. 5067-1506), libraries were pooled in equimolar amounts (6-7 libraries/pool).  The library pools were then loaded onto a HiSeq flowcell lane, and following amplification with Illumina's cBot cluster generation system, sequencing runs were performed in paired-end mode (2 x 100-bp reads) using the Illumina HiSeq2000 platform. On average, 67 million reads were generated per sample.