title: GSM3660161: MCP_12; Mus musculus; RNA-Seq
library_construction_protocol: 189 single cells were index-sorted into 2 x 96-well PCR plates containing 2.3 µl lysis buffer (2.3 U SUPERase In™ RNase Inhibitor (ThermoFisher Scientific, AM2696) in 0.2 % Triton per well), vortexed and frozen at -70 °C. Sample preparation for RNA-sequencing was performed following Picelli et al (Nature Protocols 9, 171-181 (2014)) with minor modifications. Following brief centrifugation at 2000 rpm for 2 minutes to thaw cells, oligo-dT was annealed to polyA-mRNA at 72 °C for 3 minutes. cDNA was reverse transcribed with 50 U/reaction SmartScribe Reverse Transcriptase (Takara ClonTech, 639537) without Betaine and MgCl2, and amplified using 23 PCR cycles. ERCC Spike-In Mix (Invitrogen, 4456740) was included in each single cell reaction. PCR products were cleaned using 1:0.6 ratio of cDNA: AMPure XP beads (Beckman Coulter, A63882) using BioMek FXP Dual Span Liquid Handler. Single cell cDNA library was eluted in 22 µl volume of EB Buffer (Qiagen, 19086) and 20 µl volume collected to ensure minimal bead carryover. Libraries for scRNA-sequencing were prepared using Illumina Nextera XT DNA preparation kit (Illumina, FC-131-1096) and Nextera Index Kit v2 Set A and D (Illumina, FC-121-2001 and FC-121-2004 respectively), following the manufacturer's protocol using ¼ of the recommended volumes and 1.26 µl cDNA library. Single cell libraries labelled with Index Kit A and D were pooled separately and cleaned with AMPure XP beads; first with 1:0.5 ratio of cDNA: beads then the supernatant cleaned with 1:0.3 ratio of cDNA: beads. Pooled libraries were eluted in 32.0 µl EB Buffer and 30.0 µl volume collected to ensure minimal bead carryover. Quantification was performed with KAPA Library Quant Kit (Roche, 07960140001); index kit A pooled libraries were mixed with equimolar concentration of index kit D pooled libraries and sequenced in one lane on the Illumina HiSeq4000 system (50 bp single-end reads).
attributes.tag: GEO Accession
attributes.value: GSM3660161