title: GSM8008591: Spleen, AS treated, replicate 1; Salmo salar; RNA-Seq
library_construction_protocol: Approximately 45mg of each frozen spleen sample was placed in a 6-well tissue culture plate (Stem Cell Technologies) with 1 ml TST (2 mL of 2X ST buffer + 120 µL of 1% Tween-20 + 20 µL of 2% BSA brought up to 4 ml with nuclease-free water). The tissue was minced using Noyes Spring Scissors for 10 min on ice. The resulting homogenate was filtered through a 40 µm Falcon cell strainer, and a further 1 mL of TST was added to wash the well and filter. The volume was brought up to 5 mL using 3 mL of 1X ST buffer (diluted from 2xST buffer [292 µl of 146 mM NaCl, 100 µl of 10 mM Tris-HCl pH 7.5, 10 µl of 1 mM CaCl2, 210 µl of 21 mM MgCl2, brought up to 10ml with nuclease-free water]). The sample was centrifuged at 4°C for 5 min at 500g before the resulting pellet was re-suspended in 1 ml 1X ST buffer and the recovered nuclei were filtered through a 40 µm and a 20 µm Falcon cell strainer, Hoechst stained, visually inspected under a fluorescent microscope, and counted using a Bio-Rad TC20. Spleen nuclei were processed through the 10X Chromium Single Cell Platform using the ChromiumTM Single Cell 30 Library and Gel Bead Kit v3.1 and ChromiumTM Single Cell A Chip Kit (both 10X Genomics) as per the manufacturer's protocol. For each sample, the nuclei were loaded into a channel of a Chromium 3' Chip and partitioned into droplets using the Chromium controller before the captured RNA for each nucleus was barcoded and reverse transcribed. The resulting cDNA was PCR amplified for 14 cycles, fragmented, and size selected before Illumina sequencing adaptor and sample indexes were attached.