GSM5397123: scRNA-seq Outer POD7 [18101611]; Mus musculus; RNA-Seq
Single cell RNAseq was performed via the method described by Picelli et al (42). Lysis buffer plates were thawed on ice, then heated at 72 oC/3 min in a Biorad C1000 Touch thermal cycler to denature RNA. scRNA-seq: First strand cDNA synthesis was performed in a 10 ul reaction with 100 Units of Clontech's Smartscribe reverse transcriptase (Cat. No. 639538), 10 Units RRI, 1X First Strand Buffer (Clontech), 5 mM DTT, 1M Betaine (Sigma B0300-5VL), 6mM MgCl2, 1 M Template Switch Oligo (TSO, (5′-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3′, Exiqon) at 42oC/90 min, 70oC/5 min. PCR pre-amplification was performed in a 25 ul reaction with 1X Kapa HiFi HotStart, 0.1 uM ISPCR primer (5′-AAGCAGTGGTATCAACGCAGAGT-3′, IDT) at 98oC/3 min, then 25 cycles of 98oC/20 sec, 67 oC/15 sec, 72 oC/6 min, then 72 oC/5 min. Amplified cDNAs were purified by SPRI beads using a Biomek FX automated platform and eluted in 25 ul water, and 2 ul aliquots were run on an Fragment Analyzer High Sensitivity NGS 1-6000 kit for quantitation. Barcoded sequencing libraries were made using the miniaturized Nextera XT protocol of Mora-Castilla et al(43) in a total volume of 4 ul. Pooled libraries were analyzed on an Agilent Bioanalyzer High Sensitivity DNA chip for qualitative control purposes. cDNA libraries were sequenced on a NextSeq 500 Illumina platform aiming for 50,000 reads per cell. scRNA-seq: cDNA libraries were sequenced on a NextSeq 500 Illumina platform aiming for 50,000 reads per cell.