GSM6753461: MDAMB431; CROPseq scRNAseq; Homo sapiens; RNA-Seq
After transduction, cells were grown for 7 days, then processed via 10X Genomics 3' Single Cell mRNA Capture Kit v3 according to the manufacturers protocols. Unused cDNA from the library prep was used to amplify the CRISPR sgRNA sequences to improve cell annotation. In a 50 μL reaction, 20 μL of cDNA was mixed with 2.5 μL of the CROP-Seq_Guide_Amp primer(10μM), 2.5 μL of the NEB_Universal primer (1 0μM) (Supplemental File 1), and 25 μL of Kapa HiFi HotStart ReadyMix. After amplifying and purifying the sgRNA libraries via a Qiagen PCR purification column, the libraries were then indexed for Illumina sequencing via an NEBNext multiplexed indexing oligo kit. 1 μL of the purified step 1 PCR amplicon as template (the sgRNA library) was added with 2.5 μL of each indexing primer per 50 μL Kapa HiFi reaction, and run for 6-8 cycles with an annealing temperature of 65 °C and an extension time of 45 seconds. The final sgRNA sequencing libraries were then purified using AmpureXP magnetic beads (Beckman A63881) at a 1.6:1 beads to DNA ratio.