GSM8994791: Iron; Homo sapiens; RNA-Seq
Single-cell libraries were prepared by trypsinizing and pooling replicate cultures of IMR-90 fibroblasts (n =3) exposed to 4Gy radiation and mock control. Cells were trypsinized using TrpLE (Gibco) and examined for viability (>80%) through trypan blue staining. Cells were resuspended in MEM (Gibco) supplemented with 10% FBS in 100uL at 1500 cells/uL for subsequent library preparation and sequencing provided by the University of Minnesota's Genomics Center. Library construction was done using Chromium Next GEM Single Cell 3ʹ dual index Kits v3.1 with Chip G (10x Genomics). Live suspensions of ~10,000 single cells were loaded for generation of gel bead-in-emulsions (GEMs), the captured cells were lysed, and the RNA was barcoded while reverse-transcribed in each GEM. GEMs were then broken, and the synthesized cDNA was used for library preparation following the manufacturer's protocol. cDNA quality was assessed on the Agilent Bioanalyzer with Pico-green (Agilent). The libraries underwent additional QC by shallow-sequencing using an Illumina NextSeq instrument. Final library sequencing was done using an Illumina Nova Seq S4 lane capturing ~ 20,000 paired-end reads per cell.  10x sequencing libraries were converted into feature-barcode matrices following the Cell Ranger (10x Genomics, version 5.0.0) pipeline. After demultiplexing, the function 'cellranger count' was used with default parameters, reads were mapped using the Cell Ranger prebuilt annotation platform GRCh38.