scRNA-Seq of Mus musculus: Enriched splenocytes from 3 female spleens
The 10X manufacturer's instructions found in the Chromium Single Cell' Reagents Kits v3 User Guide (CG000183 Rev A) were followed unless specified. The Roughly 10,000 enriched splenocytes (~800 cells/mL) were processed using the Chromium Single Cell 3' Reagent Kit v3 (Chromium Single Cell 3' GEM, Library & Gel Bead Kit v3), Chromium Single Cell B Chip Kit, and Chromium Controller to generate GEMs and barcoding of single cells. GEMs were transferred in tubes for GEM-RT incubations using a Bio-Rad C1000 Touch Thermocycler (thermocycling protocol: Lid temp 53C, Step 1: Temp 53C, time 45 mins; Step 2: Temp 85C, time 5 mins; Step 3: Temp 4C, time Hold). Post GEM-RT Cleanup & cDNA amplification was carried out with the adjustment of thermocycler settings containing 13 total cycles (cDNA thermocycler protocol: Lid temp 105C; Step 1: 98C, 3 mins; Step 2: 98C, 15 secs; Step 3: 63C, 20 secs; Step 4: 72C, 1 min; Step 5: Repeat from step 2 for a total # of 13 cycles; Step 6: 72C, 1 min; Step 7: 4C, Hold). cDNA cleanup using SPRIselect reagent was used per manufacturer's instructions. cDNA samples were stored at -20C for 2 weeks until cDNA QC & Quantification. cDNA QC and Quantification were performed using a Fragment Analyzer Automated CE System  12 capillaries (Advanced Analytical Technologies) using a High Sensitivity chip and subsequently analyzed using Fragment Analyzer software (Version #: 1.2.0.11; Advanced Analytical Technologies) & PROSize 3.0 (v. 3.0.1.6) Fragment Data Analysis Software (Advanced Analytical Technologies). 3' Gene Expression Library Construction was carried out per manufacturer's instructions followed by Post Library Construction QC as performed in the previous step. Single Cell 3' Gene Expression libraries were sequenced by loading 225 pM on an Illumina S1 Flow Cell using the NovaSeq 6000 S1 Reagent Kit (100 cycles), and an Illumina NovaSeq 6000 was used to sequence the library. The target sequencing depth for the 3' Gene Expression Library was 80,000 to 100,000 read pairs per cell. The sequencing read run resulted in the number of cycles: Read 1, 28 cycles; i7 Index Read, 8 cycles; Read 2, 87 cycles.