GSM4458525: 105_KO_BULGE: RNA-seq Dnmt3a KO DMBA Backskin bulge treated rep1; Mus musculus; RNA-Seq
To isolate pre-cancerous epidermal cells following short DMBA/TPA treatment, back skins were dissected and processed to single­-cell suspensions as previously described (Jensen et al. 2010).  To purify tumor cells, DMBA/TPA-induced SCCs were mechanically dissociated using a McIlwain Tissue Chopper (The Mickle Laboratory Engineering Co. LTD). Minced tumor tissue was digested under agitation in serum-free EMEM medium without calcium containing 2.5 mg/ml Collagenase I (Sigma Aldrich) and 0.75 mg/ml trypsin (Life Technologies) for 90 min at 37°C. Cells were pelleted, resuspended in 1–2 ml of 0.25% pre-warmed trypsin/EDTA (Life Technologies) containing 100 µg/ml DNase (Sigma Aldrich) per tumor, and incubated at 37°C for 2 min. Trypsin was inactivated by adding EMEM without calcium containing 10% chelated FBS. Cells were washed twice in PBS and filtered sequentially through 100 µm and 40 µm cell strainers. The libraries of total RNA from wild-type and Dnmt3a cKO tumors was prepared using the TruSeq®Stranded Total Sample Preparation kit (Illumina Inc.) according to the manufacturer's protocol. Each library was sequenced using TruSeq SBS Kit v3-HS, in paired end-mode with the read length 2 ´ 76 bp. A minimal of 137 million paired-end reads were generated for each sample run in one sequencing lane on HiSeq2000 (Illumina, Inc) following the manufacturer's protocol. Images analysis, base calling, and quality scoring of the run were processed using the manufacturer's software Real-Time Analysis (RTA 1.13.48) and followed by generation of FASTQ sequence files by CASAVA.