title: GSM9074774: Inlet 2 Forward and Reverse reads samples 5-8 (5-24h_sibmac, 6-24h_sibmac, 7-24h_mutmac, 8-24h_mutmac); Danio rerio; RNA-Seq
library_construction_protocol: All irg1-KI:GFP+ cells expressing any detectable level of GFP were isolated using FACS on a BD FACSMelody across four conditions: control uncut, control 24 hpa, control 48 hpa, and nlrc3l mutant 24 hpa. Two biological replicates were collected per condition, each consisting of pooled cells from 90 embryos, resulting in a total of eight samples. To maintain cell viability and quality, embryos were dissociated and sorted in batches of 30, with three such batches pooled to constitute a single sample of cells used for scRNA-sequencing. Cells were stained with acridine orange and propidium iodide and assessed for viability, concentration, and singleness using the LUNA-FX7 Dual Fluorescence Cell Counter (Logos Biosystems). Cells were then processed using the 10x Genomics Chromium X Controller and the Chromium GEM-X Single Cell 3' Kit v4 (PN-1000686) following the manufacturer's user guide (https://tinyurl.com/2kz2bcs4). Samples were loaded onto the GEM-X 3' Chip with a target recovery of 20,000 cells per inlet for library preparation. Single cells, reverse transcription reagents and gel beads coated with barcoded oligos were encapsulated together in an oil droplet to produce gel beads in emulsion (GEMs). Reverse transcription was performed using a C1000 thermal cycler (Bio-Rad) to generate complementary DNA (cDNA) libraries tagged with a cell barcode and unique molecular index (UMI). GEMs were then broken and purified using Dynabeads MyOne SILANE beads (Invitrogen) prior to 11 cDNA amplification cycles. Amplified cDNA libraries were purified with SPRIselect magnetic beads (Beckman Coulter) and quantified using an Agilent Bioanalyzer High Sensitivity DNA chip (Agilent Technologies). Fragmentation, end repair, A-tailing and double-sided size selection using SPRIselect beads were then performed. Illumina-compatible adapters were ligated onto the size-selected cDNA fragments. Adapter-ligated cDNA was then purified using SPRIselect beads. Uniquely identifiable indexes were added during 12 amplification cycles. The completed sequencing libraries were then purified using SPRIselect beads, visualized using the TapeStation D5000 High Sensitivity DNA ScreenTape, and pooled in an equimolar ratio. Pooled libraries were sequenced on a DNBSEQ-T7 machine (MGI Tech) at the UNC High Throughput Sequencing Facility in paired-end format (Read 1: 28 cycles, i7 index: 10 cycles, i5 Index: 10 cycles, Read 2: 90 cycles).