GSM9724316: LW662 CM Organoids, RNA; Homo sapiens; RNA-Seq
H9 WT and TBX5 +/- Organoids were generated according to prior protocols (34, 35 from Casey's paper). The organoids were harvested on D123 and single cell dissociated using trypLE for 45-60 minutes. Single cells were then loaded for library contruction Digested single cells were labelled with 12 hashtag oligo(HTO)-conjugated antibodies in 1% BSA / 50% culture media / 50% PBS, pool-washed, and resuspended in PBS with 1% BSA. Libraries were performed according to the 10X Genomics user guide CG000513 (Chromium Next GEM Single Cell 5' HT Reagent Kits v2 (Dual Index) with Feature Barcode technology for CRISPR Screening and Cell Surface Protein) with some changes. Briefly, cells were partitioned into Gel bead-in-emusion (GEMs) with Chromium X instrument. The HTO and  reverse-transcribed guide RNA and mRNA from the same GEM share the same cell barcode. Gene expression libraries, sgRNA libraries and HTO libraries were constructed from the different sized DNA fragments selected with SPRIselect reagent from amplified GEM-RT reaction mixtures. The modifications of the original 10X protocol: 1. 60k cells per library were recovered by super-loading 173k cardiomyocytes (115k neuron cells or ES cells). 2. Triple amounts of sgRNA fraction were used to construct sgRNA libraries compared to the 10X protocol.