GSM9683897: Pooled screen, 103F, v1, CD34+ enriched, GEX; Homo sapiens; RNA-Seq
10X genomics 3' transcriptome kit with v3.1 chemistry (10X genomics #1000268) processing of dissociated single cells, as per the manufacturer's instructions. All single-cell RNA-seq libraries were generated using the 10X genomics 3' transcriptome kit with v3.1 chemistry (10X genomics #1000268) as per the manufacturer's instructions. We targeted a loading concentration of 10,000 cells / lane and washed cells thoroughly in PBS-/- prior to loading to minimize background RNA contamination. We dual-indexed libraries during PCR2 using the Dual Index Kit TT (Set A; 10X genomics #1000215).  We generated corresponding single-cell TF barcode libraries by performing dial-out PCR on a sample of the 10X PCR1 product. Our primers were designed to generate a ~1.2-kb product compatible with Illumina TruSeq chemistry such that the TF barcode is sequenced by read 2 and the 10X cell barcode by read 1 (Extended Data File 5). The forward primer anneals to the 10X PCR2 adapter sequence upstream of the 10X cell barcode and adds an Illumina p5 adapter, 8-bp barcode, and TruSeq R1 primer annealing site. The reverse primer anneals immediately 5' of the 24-bp TF barcode and adds an Illumina p7 adapter, 8-bp barcode, TruSeq R2 primer annealing site, and 12-bp stagger to increase sequence diversity. Amplification was performed using NEBNext HiFi polymerase mix (NEB #M0541L). 5% of the total 10X PCR1 product was used template – i.e., 2 uL containing ~50 ng DNA. Primers were used at a 0.25 uM final concentration. Cycling conditions were: 1. 98°C 3 minutes; 2. 20 x [98°C 10 seconds, 60°C 10 seconds, 72°C 1 minute]; 3. 72°C 1 minute. PCR product was purified for sequencing with a 0.8X SPRI selection (Beckman Coulter #B23317). Successful amplification and purification of the expected 1.2-kb product was verified by High-Sensitivity DNA Analysis (Agilent #5067-4626).  Single-cell transcriptome libraries were pooled and sequenced on an Illumina NextSeq. We typically pooled up to 40,000 cells per sequencing run and targeted 20,000 reads/cell sequencing depth. We used High-Output 500/550 v2.5 75-cycle kits with the following read distribution: R1: 28, R2: 44, i7: 10, i5: 10. Corresponding single-cell TF barcode libraries were pooled and sequenced similarly, but with the following read distribution: R1: 28, R2: 53, i7: 0, i5: 8.