GSM3664151: CD34_Neg_Basophil_18; Mus musculus; RNA-Seq
94 single cells were index-sorted into a 96-well PCR plate 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)) and GSE128003 with modifications. Following brief centrifugation at 2000 rpm to thaw cells, oligo-dT was annealed to polyA-mRNA at 72 °C for 3 minutes. cDNA was reverse transcribed with 20 U/reaction Maxima H Minus Reverse Transcriptase (ThermoFisher) in the presence of PEG8000 (Sigma), and amplified using 19 PCR cycles with 1.25 U Terra PCR Direct Polymerase per reaction (Takara). 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). Single cell cDNA library was eluted in 26 µl volume of EB Buffer (Qiagen, 19086) and 25 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 D (FC-121-2004), following the manufacturer's protocol using ¼ of the recommended volumes and 1.25 µl cDNA library. Single cell libraries were pooled 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 25 µl EB Buffer and 23 µl collected to ensure minimal bead carryover. The libraries were sequenced on the Illumina HiSeq4000 system (50 bp single-end reads).