GSM5387658: CROP-Seq Day 8 iTF-Microglia sgRNA enrichment Lane 03; Homo sapiens; OTHER
For QuantSeq, cell culture medium was aspirated, cells were washed once with DPBS, and RNA lysis buffer was added directly to wells containing either Day 0 iTF-iPSCs, Day 9 iTF-Microglia or Brownjohn-iMG +/- 100 ng/ml 24h LPS treatment, or Day 15 iTF-Microglia. Biological triplicates for each condition (approximately 0.15 Mio cells each) were pelleted, snap frozen, and stored at -80°C. RNA was extracted using the Quick-RNA Miniprep Kit (Zymo; Cat. No. R1055). For CROP-seq, Day 8 iTF-Microglia were washed 3X with DPBS, dissociated with TrypLE, and resuspended in nuclease-free water before loading onto four wells of the 10x Chromium Controller (10x Genomics, v3.1) according to the manufacturer's protocol, with 35,000 cells recovered per sample as the target.  For QuantSeq, libraries were prepared from total RNA (473 ng per sample) using the QuantSeq 3' mRNA-Seq Library Prep Kit for Illumina (FWD) (Lexogen; Cat. No. 015UG009V0252) following the manufacturer's instructions. Library amplification was performed with 14 total PCR cycles. mRNA-seq library concentrations (mean of 1.13 ± 0.66 ng/uL) were measured with the Qubit dsDNA HS Assay Kit (Invitrogen; Cat. No. Q32851) on a Qubit 2.0 Fluorometer. Library fragment-length distributions (mean of 287 ± 28 bp) were quantified with High Sensitivity D5000 Reagents (Agilent Technologies; Cat. No. 5067-5593) on the 4200 TapeStation System. The libraries were sequenced on an Illumina NextSeq 2000 instrument with single-end reads. For CROP-seq, sample preparation was performed using the Chromium Next GEM Single Cell 3′ Reagent Kits version 3.1 (10x Genomics, cat. no. PN-1000121) according to the manufacturer's protocol, reserving 10-30 ng full-length cDNA to facilitate sgRNA assignment by amplifying sgRNA-containing transcripts using hemi-nested PCR reactions adapted from a previously published approach (Hill et al., 2018; Tian et al., 2019). cDNA fragment analysis was performed using the 4200 TapeStation System and sgRNA enrichment libraries were separately indexed and sequenced as spike-ins alongside the whole-transcriptome scRNA-seq libraries using a NovaSeq 6000 using the following configuration: Read 1: 28; i7 index: 8; i5 index: 0; Read 2: 91. 