alias: GSM2048612
title: GSM2048612: CSLKO2-78; Homo sapiens; RNA-Seq
library_construction_protocol: Tumors with surrounding stromal tissues from xenografts were minced and nonenzymaticallydigested using cell dissociation enzyme-free PBS-based (Gibco) for 30 minutes at 37o C withoccasional pipetting. The dissociated cells were then filtered through a 40 mm nylon mesh strainer. Single cellsuspension was obtained by re-suspension in CO2 -independent medium (Gibco) for single cell picking. Singlecells of xenografts were manually picked under the microscope as previously described (Picelli et al., 2013).Briefly, a single cell was picked using mouth pipette under 20X magnification in 0.5 ml solution. The pickedsingle cell was released into RNase-free PCR strip tube containing hypotonic Smart-seq RNA lysis bufferconsisting of 2.3 ml of 19:1 ratio of 0.4% TritonX-100 and RNase inhibitor (Clontech), 1 ml of 10 mM dNTP(Fermentas), 1 ml of 10 mM oligo-dT primer, and ERCC spike-in (Ambion) at 72o C for 3 min. Total RNA from tumors and cells was purified using the RNeasy Mini  Kit(QIAGEN), according to the manufacturer’s instructions. RNA concentration and RNA integrity number (RIN)were calculated by using the Agilent RNA 6000 Nano  Kit (Agilent Technologies). Two biological replicates for each RNA sample were processed to cDNA library preparations using the Illumina TruSeq™ RNA sample preparation  kit (Low-Throughput protocol) according to the manufacturer’s protocol. Quantification and qualitycontrol of the cDNA libraries were done using the Agilent DNA 1000 Kit (Agilent Technologies). The libraries were sequenced on an Illumina HiSeq 2000 system.
identifiers.namespace: GEO
identifiers.id: GSM2048612