GSM8864215: Pediatric day 14 untreated; Homo sapiens; RNA-Seq
For the tissue sections that received barcoded viral libraries (total titer 2.1E9 in 4 µL PBS per tissue slice), 14 days after virus application the tissue was gently removed from its well, flash frozen in liquid nitrogen, and stored at -80 oC. On the day of processing, frozen tissue was dissociated using GentleMACS nuclei isolation protocol (nuclei isolation buffer [Miltenyi Biotec, cat# 130-128-024], Protector RNAse Inhibitor [Millipore Sigma, cat# 3335402001], GentleMACS C tubes [Miltenyi Biotec, cat# 130-093-237], GentleMACS Octo Dissociator [Miltenyi Biotec, cat# 130-096-427], MACS SmartStrainers 70um [Miltenyi Biotec, cat# 130-098-462], MACS SmartStrainers 30um [Miltenyi Biotec, cat# 130-098-458]). In brief, samples were placed in 2 mL of Miltenyi Nuclei Isolation Buffer and Protector RNAse Inhibitor in GentleMACS C tubes. Samples then underwent the preprogrammed “nuclei isolation” program on a GentleMACS Octo Dissociator. Immediately after dissociation, samples were strained through a 70um MACS SmartStrainer and collected in a 15 ml tube, centrifuged at 300xg for 5 minutes at 4°C. The supernatant was extracted and discarded, and the resulting pellet resuspended in 1mL of ice-cold PBS. Resuspended samples were then run through a 30um MACS SmartStrainer, centrifuged, and resuspended in ~250-500 µL. Nuclei were then FACS sorted using DAPI on a Sony MA900 in the Baylor Cytometry and Cell Sorting Core. The single-cell gene expression libraries were prepared according to the Chromium Single Cell Gene Expression 3'v3.1 instruction (PN-1000121, PN-1000120, PN-1000213, 10x Genomics). Briefly, single cells, reverse transcription (RT) reagents, Gel Beads containing barcoded oligonucleotides, and oil were loaded on a Chromium controller (10x Genomics) to generate single-cell GEMS (Gel Beads-In-Emulsions) where full-length cDNA was synthesized and barcoded for each molecule of mRNA (UMI) and each single cell (cell barcode). Subsequently, the GEMS were broken and cDNA from each single cell was pooled. Following cleanup using Dynabeads MyOne Silane Beads, cDNA is amplified by PCR. The amplified product was fragmented to optimal size before end-repair, A-tailing, and adaptor ligation. The final library was generated by amplification.
GEO
GSM8864215_r1