title: GSM2858620: GMP_12; Mus musculus; RNA-Seq
library_construction_protocol: Single cells were sorted into 96 well PCR plates containing 2.3 µl lysis buffer consisting of 2.3 U SUPERase In™ RNase Inhibitor (ThermoFisher Scientific, AM2696) in 0.2 % Triton per well. Cells were processed for RNA-sequencing following Picelli et al (Nature Protocols 9, 171-181 (2014)). Briefly, oligo-dT was annealed to polyA-mRNA at 72 °C for 3 minutes, followed by cDNA synthesis using SuperScript™ II Reverse Transcriptase (Invitrogen 18064014). PCR was performed with 23 PCR cycles and products cleaned up using 1:0.6 ratio of cDNA: AMPure XP beads (Beckman Coulter). ERCC Spike In Mix was included in each single cell reaction at 1: 3 000 000 dilution (ThermoFisher Scientific 4456740). Single cell cDNA library was eluted in 20 µl volume of EB Buffer (Qiagen). Prior to library preparation, cDNA library was checked on Agilent Bioanalyser 2100 with Agilent High Sensitivity DNA Kit. Library preparation was performed using Illumina Nextera XT DNA Library Preparation Kit with 1.26 µl cDNA library and Nextera XT Index Kit v2 Set A and D. Single cell libraries were pooled for all indexed samples and cleaned with AMPure XP beads; first with 1:0.5 ratio of cDNA: beads then the supernatant cleaned with 1:0.2 ratio of cDNA: beads.   Pooled libraries were quantified with KAPA Library Quant Kit (KAPA Biosystems KK4824) and single-end 50 base pair sequencing was performed on the Illumina HiSeq 4000 platform.
attributes.tag: GEO Accession
attributes.value: GSM2858620