GSM7059704: wtgl6.3077-biol rep 2; Mus musculus; RNA-Seq
Primary tissues (tumor or gland) were isolated from mammary by stepwise mechanical disruption and enzymatic digestion according to our published protocols. Tissues (tumor or gland) were harvested from 6- to 10-wk-old mice, minced with a scalpel, and incubated for digestion for 2 h at 37 °C with gentle rocking in FBS free DMEM (Multicell, Wisent Inc., St. Bruno, Quebec, Canada) supplemented with 2.4 mg/ml collagenase B and 5U/ml Dispase II (4942078001, Roche, Switzerland). Tissue fragments were washed with PBS, centrifuged, and resuspended in DMEM with 10 % Fetal Bovine Serum (Gibco, US Origin), 100 IU/mL penicillin and 100 ug/mL streptomycin (Multicell, Wisent Inc., St. Bruno, Quebec, Canada). Ribosomal RNA (rRNA) depletion was achieved using an Illumina Ribo-Zero Magnetic kit. First strand cDNA synthesis used Super strand synthesis Act D kit containing random primers and Super Script II reverse transcriptase to convert the original mRNA to complementary cDNA. Second strand cDNA synthesis involves removal of the original RNA template and synthesizes a replacement strand, incorporating dUTP in place of dTTP to generate ds cDNA. After cDNA synthesis, a single “A” nucleotide is added to the 3' ends of the blunt fragments to prevent them from ligating to one another during the adapter ligation reaction. During adapter ligation process, each sample has a different RNA adapter index for the identification in subsequent data analyses (Illumina® Nextera™ DNA Unique Dual Indexes). AMPure XP beads were used to purify the ds cDNA from the second strand reaction mix and leaving exclusively the blunt-ended cDNA for the subsequent DNA enrichment. The PCR was performed with a PCR primer cocktail that anneals to the ends of the adapters in order to exclusively enrich and amplify those DNA fragments that have adapter molecules on both ends. Only the first strand cDNAs were amplified in this step. Quantification by LightCycler® 96 Instrument with KAPA library quantification kit. The RNAseq transcriptome strand library was prepared by following the TruSeqTM Stranded Total RNA kit from Illumina (San Diego, CA, USA) using 100 ng of total RNA for each sample. Paired-end RNA-seq library was sequenced with Illumina NovaSeq6000 at McGill Genome Center according to the standard protocol.