GSM8946289: Spleen, 5prime, modified protocol, long-read; Mus musculus; RNA-Seq
Spleens were isolated from mice under ketamine/xylazine/acepromazine anesthesia. Spleens were dissociated through a 70 µm strainer in cIMDM using a 3 mL syringe plunger. Cells were washed, centrifuged (400 × g, 5 min), and treated with 1 mL ACK lysis buffer for 30 seconds, followed by dilution in cIMDM and a second spin. After one additional wash, cells were resuspended in 6 mL FACS buffer. Viable cells were counted by trypan blue exclusion. Single-cell 3' libraries were prepared using the Chromium Next GEM Single Cell 3ʹ Kit v3.1 following the protocol up to and including step 2.4, stopping just before fragmentation. Single-cell 5' libraries were prepared using the Chromium Next GEM Single Cell 5' Kit v2  following the protocol up to and including step 2.4, stopping just before fragmentation.  For the optimized libraries, the following modifications were applied to the 5' library preparation: 1 ul 10 mM dNTP solution (Thermo Scientific FERR0191) was added to the reaction in step 1.1. The extension time was increased from 45 minutes to 2 hours in step 1.5. 1 ul 10 mM dNTP solution was added to the reaction in step 2.2 and the extension time was increased from 1 minute to 3 minutes. Long-read libraries were prepared from the cDNA using Sequencing Kit V14 (Nanopore SQK-LSK114) and the PCR Expansion (Nanopore EXP-PCA001). For 3' libraries, the Ligation sequencing V14 — single-cell transcriptomics with 3' cDNA prepared using 10X Genomics on PromethION (SQK-LSK114) protocol was used.  For 5' libraries, the Ligation sequencing V14 - Single-cell transcriptomics with 5' cDNA prepared using 10X Genomics on PromethION (SQK-LSK114) protocol was used. Short Fragment Buffer (SFB) was used for library preparation instead of Long Fragment buffer (LFB). Library Beads (LIB) were used for the flow cell priming mix stead of Library Solution (LIS). Libraries were sequenced on R10.4.1 flow cells on either a PromethION 2 Solo (P2S) or PromethION  2 Integrated (P2i).