GSM8275374: cKO1_cortex; Mus musculus; RNA-Seq
Cell suspensions are handed off to the Broad Clinical Labs Single Cell processing team. Appropriate volume adjustments are made to ensure thetarget cell recoveryis met prior to the addition of reverse transcription mastermix. The cells in mastermix are then added to the 10x chip G along with 10x gel beads and partitioning oil. The chip is run on the 10x Chromium Controller to create an emulsion of single cells, RT mastermix and a barcoded gel bead. After emulsions are created, samples are immediately put on reverse transcription thermalcycler protocol set at 53OC for 45 minutes, 85OC for 5 minutes before holding at 4OC. The emulsion is then broken, and debris is removed via a Silane bead cleanup. The purified cDNA is then amplified with the following PCR conditions: Initial denaturation at 98°C for 3 minutes, followed by12 cyclesof denaturation at 98°C for 15 seconds, annealing at 63°C for 20 seconds, and extension at 72°C for 1 minute. A 0.6X SPRI cleanup is performed before being quanted on a Qubit. cDNA library construction is run on an entirely automated workflow. The cDNA is fragmented and repaired with the addition of an A-tail at 32OC for 5 minutes then 65OC for 30 minutes. A 0.8X double sided SPRI cleanup then removes undesired fragments. Adapters are ligated onto the DNA at 20OC for 15 minutes followed by a 0.8X SPRI. 10x dual indexed barcodes are added before a PCR reaction with the following conditions: An initial denaturation at 98°C for 45 seconds, followed by 12 cycles of denaturation at 98°C for 20 seconds, annealing at 54°C for 30 seconds, and extension at 72°C for 20 seconds. These libraries undergo a final 0.8X double sided SPRI cleanup for size selection. Following PCR Cleanup, quality is confirmed using Quant-iT PicoGreen and an Agilent TapeStation to confirm library size. Samples are pooled together to accommodate the desired reads per cell.