title: Sample 3
organism: Gadus morhua
description: Protocols: Two to ten million single spleen cells from each fish were resuspended and homogenized in RLT lysis buffer (Qiagen) and kept frozen at –70°C until RNA isolation.  Total RNA was purified from the spleen cells using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's instructions. First strand cDNA was made from approximately 500 ng total RNA in the presence of a template-switch oligo (TSO) that enabled the incorporation of a universal priming site at the 3'-end of the first strand cDNA. The first-strand synthesis reaction consisted of 1 µM oligo-dT primer (all primer sequences are found in Supplementary table 1), 1 mM dNTP, 1x RT buffer, 2.5 mM DTT, 8 mM MgCl2, 1 µM betaine (Merck), 0.8 U/µL Murine RNase Inhibitor (NEB), 1 µM TSO, and 8 U/µL Superscript II (Invitrogen) in a total volume of 40 µL. The reaction mix was incubated for 90 min at 42°C, followed by an inactivation step of 72°C for 15 min. In the first of total three semi-nested PCR reactions, 1.5 µL first strand cDNA was used as template where both Ig heavy and Ig light chains were targeted in the same PCR reaction. The first PCR reaction contained also 40 nM of STRTfwd_long primer, 200 nM each of the primers STRTfwd_short, IGHC_O, IGLC_O1, IGLC_O2, IGLC_O3, 200 µM dNTP, 1x Phusion high-fidelity buffer, 0.1 µL of Phusion polymerase (ThermoFisher) in a total reaction volume of 15 µL, and run with the cycling condition of denaturation 1 min at 98C; five cycles of (10s at 98°C, 60s at 72°C); five cycles of (10s at 98°C, 30s at 70°C, 40s at 72°C); eight cycles of (10s at 98°C, 30s at 68°C, 40s at 72°C); and final extension of 4 min at 72°C. In the following second PCR, the Ig heavy and light chains were amplified in separate reactions each with 1 µL product from the first PCR reaction as template. In a total volume of 10 µL, the second PCR reactions contained 40 nM of STRTfwd_long primer, 200 nM each of the primers STRTfwd_short and IGHC_M (for Ig heavy chain second PCR); or IGLC_M1, IGLC_M2, IGLC_M3 primers (for Ig light chain second PCR); 1x KAPA HiFi HotStart ReadyMix (Roche), and run with the cycling condition of denaturation of 2 min at 95°C; 10 cycles of (15s at 98°C, 30s at 65°C, 40s at 72°C); and final extension of 5 min at 72°C. In the third PCR, 1 µL product from the second PCR reaction was used as template in each of the four third PCR reactions, one for Ig heavy chain and three reactions for the three Ig light chain subclasses. In a total volume of 15 µL, each third PCR reaction contained 200 nM each of the primers R2_STRT_bulk and IGHC_I (for Ig heavy chain); or IGLC_I1, IGLC_I2, IGLC_I3 primers (for Ig light chain) and run with the same KAPA polymerase and cycling condition as the second PCR reactions. Each of the primers used in the third PCR contains a unique 6-nt barcode that identifies the sample origin, as well as portions of oligo sequences that are necessary for Illumina sequencing platforms. In the final library PCR reactions, 1.5 µL product from the third PCR reaction was used as template in total 20 µL reaction containing 200 nM each of the R1_library and R2_library primers containing sequences that completed Illumina sequencing adapters, 1x KAPA HiFi HotStart ReadyMix and run with the cycling condition of denaturation 2 min at 95°C; 10 cycles of (15s at 98°C, 30s at 60°C, 40s at 72°C); and final extension of 5 min at 72°C.