Illumina HiSeq 2000 sequencing; GSM2706561: WG960011_A02-W01; Mus musculus; RNA-Seq

WG960011_A02-W01

We adopted the STRT-seq [Islam et al., Nat Methods 11, 163-166 (2013)] RNA-seq technology to a 9600-well array and applied it to analyze single cells from mouse and human cortex single cells. Overall design: 2192 single cells from mouse somatosensory cortex and 2028 single nuclei from human post-mortem middle temporal gyrus cortex.

A novel addressable 9600-microwell array single cell RNA-seq method applied on fresh mouse cortical cells and frozen human cortical nuclei

Submitted by Gene Expression Omnibus on 07-DEC-2017

Mice were anesthetized with isoflurane, perfused with ice-cold aCSF, brains collected and sectioned using a vibratome or brain matrix and the somatosensory cortex was microdissected. Single cell suspensions were generated using the Worthington Papain dissociation system. Human nuclei were isolated from a -80°C frozen tissue piece using Dounce homogenization. 50nl lysis mix (500nM STRT-P1-T31, 4.5nM dNTP, 2% Triton-X-100, 20mM DTT, 1.5U/μl TaKaRa RNase Inhibitor) was dispensed, followed by 3min lysis at 72°C. Then, 85nl reverse transcription (RT) mix (2.1X SuperScript II First-Strand Buffer, 12.6mM MgCl2, 1.79M betaine, 14.7U/μl SuperScript II, 1.58U/μl TaKaRa RNase Inhibitor, 10.5μM P1B-UMI-RNA-TSO) were dispensed and RT carried out 42°C for 90 minutes. 96 Transposome stocks were assembled (6.25μM barcoded adapter (STRT-Tn5-Idx[1-96]), 6.25μM Tn5 transposase, 40% glycerol), 37°C 1h. Tn5 reactions were assembled with 3μl transposome and 2μl amplified cDNA, in a total 20μl 1x CutSmart buffer (NEB), and incubated at 55°C 20min.

A novel addressable 9600-microwell array single cell RNA-seq method applied on fresh mouse cortical cells and frozen human cortical nuclei

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