title: GSM8559465: Fractionated spleen, Biological replicate 2; Ictalurus punctatus; RNA-Seq
library_construction_protocol: Three channel catfish (~ 1 kg) were euthanized in a solution of buffered MS-222 for 10 min. The individual spleens were removed and placed into Petri dishes containing Leibovitz's L-15 medium adjusted to catfish tonicity (cL-15) with PenStrep glutamine solution and 5% FBS.  The spleens were first processed through 70 µM and then 40 µM cell sieves into a 50 mL Falcon tube and cL-15 was added up to 10 mL. The splenic cell suspensions were washed 3 times in 10 mL cL-15 and centrifuged at 1200 RPM for 5 min. The final cell pellet was resuspended in 15 mL cL-15. Cells were counted, and cell viability was assessed. ~ 1.0x107 cells per sample were pelleted and resuspended in 1 mL of freezing medium (90% FBS, 10% DMSO) for another project. The remaining unfractionated cells were placed over a Ficoll density gradient and centrifuged at 1200 RPM for 15 minutes, no brake. The buffy coat was removed and washed one time and then stained with recombinant 9E1 mAb (anti-catfish IgM) and then labeled with a goat anti-mouse IgG FITC Ab. 1-3 x106 IgM+ cells were sorted using a Beckman Coulter CytoFLEX SRT cell sorter using lymphocyte and FITC gates. Sorted cell samples were pelleted and resuspended in 1 mL of freezing medium (90% FBS, 10% DMSO). All cell samples were placed into Mr. Frosty freezing containers at -80°C for 24 h. The cryogenic vials were then moved into a liquid nitrogen storage until be shipped to the sequencing vendor. Frozen cells were thawed in a water bath set to 28°C and added to 6 mL of cL-15. The cells were centrifuged at 1200 RPM for 5 minutes. The supernatant was removed, and the cells were resuspended with 1 mL of cl-15. Approximately 2.5 x 106 cells were transferred to new tubes, centrifuged at 600 RPM for 5 minutes at 4°C, and the supernatant was removed. The cells were lysed in 200 µl of lysis buffer and incubating for 1 minute on ice. The lysis buffer consisted of Tris-HCL at pH 7.4, NaCl, MgCl2.  The lysed cells were washed with 800 µl of buffer solution and centrifuged. The buffer contained BSA Solution, RNase Inhibitor, and 1x PBS. The nuclei were washed two times using 1 mL of buffer solution, pelleted and the supernatant was removed. Centrifugation was carried out at 1200 RPM for 10 minutes at 4°C. The nuclei concentration was diluted to the target concentration required for library preparation. The single-nuclei RNA-seq libraries were prepared and constructed using the Chromium X Instrument (10x Genomics) and the Chromium Next GEM Single Cell 3' GEM Kit v3.1 (10x Genomics) following the manufacturer's protocol. The individual nuclei were partitioned to produce gel beads-in-emulsion (GEMs). The GEMs are generated by loading the nuclei in master mix, barcoded Single Cell 3' v3.1 Gel Beads and partitioning oil onto Chromium Next GEM Chip G. The Gel Bead was dissolved to release the primers and incubated to produce barcoded full-length cDNA. The cDNA was cleaned to removed left over reagents using Dynabeads MyOne SILANE (ThermoFisher Scientific, Waltham, MA 2000048), then amplified via PCR. Optimal sized cDNA amplicons were selected using SPRIselect (Beckman Coulter, Brea, CA, B23318) and Illumina indexes and adapters were added via end repair, A-tailing, adaptor ligation, and PCR. Sample quality was checked using a TapeStation (Agilent, Santa Clara, CA) and TapeStation High Sensitivity D1000 ScreenTape (Agilent, Santa Clara, CA). cDNA samples were multiplex and sequenced using the Illumina NovaSeq X Plus sequencer (San Diego, CA).