GSM5287004: GTGT13; Mus musculus; RNA-Seq
Tumors were minced and digested in RPMI 1640, 1 mg/ml Collagenase II (Sigma-Aldrich), 50 µg/ml DNaseI (Roche), and 0.1% (wt/vol) BSA for 30 min at 37°C with 750 rpm agitation. Cell suspensions were subsequently passed through a 70-µm cell strainer (BD Biosciences) and collected by centrifugation. Purification of total RNA from sorted cells was performed by using the RNeasy Plus Micro Kit (Qiagen) and concentration was determined using the Quant-IT RiboGreen RNA assay kit (Thermo Fisher Scientific). Full length cDNA were generated from 1000 pg of total RNA using Clontech SMART-Seq v4 Ultra Low Input RNA kit for Sequencing (Takara Bio Europe, Saint Germain en Laye, France) according to manufacturer's instructions with 12 cycles of PCR for cDNA amplification by Seq-Amp polymerase. Six hundreds pg of pre-amplified cDNA were then used as input for Tn5 transposon tagmentation by the Nextera XT DNA Library Preparation Kit (96 samples) (Illumina, San Diego, CA) followed by 12 cycles of library amplification. Following purification with Agencourt AMPure XP beads (Beckman-Coulter, Villepinte, France), the size and concentration of libraries were assessed by capillary electrophoreris.