GSM9241583: Islet, 5prime, insulin-depleted, modified protocol, long-read, rep2; Mus musculus; RNA-Seq
Pancreatic islets were isolated from mice under ketamine/xylazine/acepromazine anesthesia by collagenase delivery into the pancreas via injection into the bile duct. The collagenase-inflated pancreas was surgically removed and digested. After isolation, islets were dissociated using Accutase in a 37°C bead bath for 25-30 minutes. Single-cell suspension was filtered through a 40 mm filter and quenched in RPMI media with 10% FBS. Single-cell suspensions were loaded into a Genomics Chromium targeting 4000 cells per sample. 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. Insulin depletion was performed on cDNA from step 2.4 of the 10X Genomics Chromium library preparation using the DepleteXTM RNA Depletion Panel (Insulin) kit from Jumpcode Genomics. We followed the PacBio MAS-IsoSeq protocol (December 2022, Version 1.0) with the following modifications: During RNP Complex Formation (Step A), we used 0.9 ul Cas9 instead of 2.3 ul, and 1.6 ul Insulin Guide RNA instead of 4.0 ul Single Cell Boost Guide RNA. During Bead Cleanup (Step D), we used 50 ul (1X) AMPure XP Beads instead of 75 ul 1.5X SMRTbell Cleanup Beads. 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).