title: GSM5141510: Untreated cell 48 Bio Rep 2; Mus musculus; RNA-Seq
library_construction_protocol: Dissected tumors were finely chopped with scalpels and the obtained fragments digested in HBSS (with Calcium and Magnesium; GIBCO) containing a mix of Collagenases 1 and 3 (Worthington; 3 mg ml−1), dispase II (Roche; 7 mg ml−1), DNase I (Invitrogen; 2 U ml−1) and left at 37 °C with gentle agitation for 20 minutes. A total of 25x106 cells were stained in a final volume of 100 μl of antibody mix for 15-20 minutes, in ice, protected from light. Cells were subsequently washed with 2 ml PBS-5% FBS, centrifuged at 500g for 5 minutes and resuspended in 500 μl PBS-5% FBS. 7-AAD was added right before the sample acquisition. Sorting was performed by using a Fusion cell sorter (BD Biosciences). The dissociated cells were centrifuged at 500g for 5 minutes, resuspended in PBS-5% FBS and filtered through 50 μm cell strainers before being counted and used for flow cytometry. 100 cells per population were sorted in 4 μl of lysis mix (0.4% Triton X + RNAse Inhibitor (1:20), dNTPS (10 mM), Oligo dT (10 μM) - according to the original Smart-seq2 protocol49) for bulk RNAseq, and 1 cell per well of a 96 well plate for single cell RNAseq, and immediately processed for RNA-sequencing or stored at -80 °C. A 5.7 μl of retro-transcription mix (see reference for details) was added to each sample. Retro-transcription was carried out according to the original Smart-seq2 protocol and cDNA was then pre-amplified for 15 cycles. After PCR pre-amplification, cDNA was purified using Ampure XP magnetic beads according to the manufacturer's instructions, in a ratio of 0.8 to 1 with cDNA, resuspended in 17.5 μl of buffer EB (Qiagen) and stored at –20 °C. Quality and concentration of the cDNA generated were assessed using High-Sensitivity Bioanalyzer kit (Agilent). 1 ng of pre-amplified cDNA was tagmented and indexed with Nextera XT DNA Sample Preparation kit (Illumina) according to the manufacturer's instructions. The product was purified with AMPure XP beads (1:1 ratio) and eluted in 17.5 μl of EB buffer (Qiagen). Samples were loaded on a High-Sensitivity DNA chip (Agilent Technologies) to check for library size and quality and concentration measured with Qubit High-Sensitivity DNA kit (Invitrogen). Libraries were pooled to a final concentration ranging between 2 nM and 10 nM and sequenced with Illumina Hiseq 4000 (25 bp single-end read) at the Wellcome Trust Centre for Human Genetics in Oxford.
attributes.tag: GEO Accession
attributes.value: GSM5141510