GSM9629623: Control replicate 2; Gallus gallus; RNA-Seq
Approximately 10 cm of ileum was excised, flushed with PBS, and opened longitudinally. The mucosa was scraped, resuspended in PBS with 5% chicken serum, 2 mM EDTA, and 2 mM DTT, and shaken at 37C/150 rpm for 45 min. Supernatant was passed through gauze, resuspended in 40% Percoll, and overlaid on 70% Percoll for density gradient centrifugation. IEL fractions from 8 birds were pooled into 4 samples per group (2 birds/pool). Samples were filtered through 40 um strainers and enriched for CD45+ cells by MACS (CD45-biotin antibody, Southern Biotech). For scRNA-seq, 4 pooled samples per group were further combined in pairs to yield 2 scRNA-seq samples per group. Enriched CD45+ IELs were washed and resuspended in PBS + 5% FBS. Approximately 5000-13000 cells per sample were loaded onto the 10x Genomics Chromium X using the GEM-X Universal 3' Gene Expression v4 kit. Libraries were constructed using 10x Genomics Library Construction Kit C and sequenced on an Illumina NovaSeq X Plus at Admera Health (South Plainfield, NJ) with 2x150 bp paired-end reads at ~70,000 reads/cell.