GSM8655815: Intestinal enteroids, D0, Layer, replicate 2; Gallus gallus; RNA-Seq
The villi were dissociated into single cells for fluorescence-activated cell sorting (FACS). Dissociation was performed using 0.25% Trypsin-EDTA solution (1X) (Gibco, UK) for 15 min at 37°C, with gentle pipetting up and down every 5 minutes. The reaction was stopped with DMEM containing 10% FBS (Gibco, UK). Single cells from the dissociated villi were washed twice in PBS supplemented with 1% BSA (Sigma-Aldrich, UK) at 400 x g for 5 min (at 4°C) and resuspended in PBS with 1% BSA and 0.4 U/μL of Protector RNase inhibitor (3335399001, Roche; collection buffer). At day 3 the organoids were dissociated using the same procedure as used for the villi. Cell viability and counts were assessed using a hemacytometer. The cells were maintained on ice before FACS was performed using a Bigfoot Cell Sorter (Thermo Fisher Scientific, UK) to obtain a population of live, single cells. Cells were collected in low retention 1.5 ml Eppendorf tubes containing 100 μL of collection buffer. scRNA-seq libraries were prepared by the Roslin Institute Genomics Platform Facility, using the 10X Genomics Chromium Next GEM Single Cell 3' Kit (v3.1) following the manufacturer's protocol. FACS sorted cells for eight samples (n=2 per broiler and layer at Day 0 and Day 3) were re-counted using a hemacytometer and diluted to 700-1,200 cells per µL. Each sample was loaded onto a Chromium Chip G lane targeting 5,000 cells per sample. After encapsulation by gel beads in emulsion (GEMs), the mRNA was captured, barcoded and reverse transcribed, before the emulsion was broken and the cDNA pooled, purified and amplified using 12 PCR cycles. The cDNA was fragmented, end-repaired and size-selected to optimise amplicon size, followed by adaptor ligation and sample indexing (11 PCR cycles). The resulting libraries were pooled in equimolar concentrations and sequenced on an Illumina NextSeq 2000 using NextSeq 1000/2000 P3 Reagents (100 cycles) v3 Kit, over two P3 flow cells. PhiX Control v3 library was spiked at a concentration of ~1%.