GSM4160556: Autopsy NEUN+ TL Control sample 10662; Homo sapiens; RNA-Seq
1) Single cell RNA-seq, TLE tissue from five different de-identified patients was obtained from the operating room and immersed in freshly prepared live cell buffer for single cell dissociation.    2) For NEUN+/PAX6+/OLIG2+ RNA-seq, 200-500mg of frozen tissue was manually homogenized in a hypotonic lysis buffer (0.32M Sucrose/5mM CaCl2/3mM Mg(Ac)2/0.1mM  EDTA, 10mM Tris-HCL pH8/1mM DTT/0.1% Triton X-100). Nuclei were collected in this buffer and purified  from cellular debris by ultracentrifugation in a sucrose gradient, resuspended in 1X PBS, simultaneously incubated with fluorescently-conjugated primary antibodies to NEUN, OLIG2, and PAX6, and isolated by FACS. 3) For EGFR+ RNA-seq, fresh tissue was  enzymatically dissociated with papain, debris was removed by passage through 22% Percoll, and dissociated cells were incubated for 30 min on ice with EGF-APC for positive selection of EGF-bound (i.e. EGFR+) populations and with  inflammatory and endothelial cell markers for exclusion: CD45-PE, CD34-PE, CD24-PE. EGFR+CD34-CD45-CD24-DAPI- cells were isolated by FACS. 1) Single cell RNA-seq was performed using the Chromium platform (10x Genomics) with the 3' gene expression (3' GEX) V1-V3 chemistry, using an input of ~10,000 single cells. Gel-Bead in Emulsions (GEMs) were generated on the sample chip in the Chromium controller.   2) For NEUN+/PAX6+/OLIG2+ RNA-seq library preparation (SMARTer Stranded Total RNA-Seq Kit Pico Input  Mammalian), 2952pg of total nuclear RNA were amplified into cDNA with  fragmentation times of 2.5 min for autopsy and 3.5 min for TLE cases (14 cycles total amplification for all).  Ribosomal RNA depletion was performed using human-specific R-Probes. 3) For EGFR+ RNA-seq library preparation  (SMART-seq v4, Clontech Laboratories), 165-234pg total RNA was converted to cDNA (8-13 cycles).  Libraries were generated using Nextera XT (FC-131-1024) and validated using Agilent 2100 Bioanalyzer. 1) For single cell RNA-seq, Barcoded cDNA was extracted from the GEMs by Post-GEM RT-cleanup and  amplified for 12 cycles. Amplified cDNA was fragmented and subjected to end-repair, poly A-tailing, adapter  ligation, and 10X-specific sample indexing following the manufacturer's protocol.
GEO Accession
GSM4160556