GSM5848677: RD cell line; Homo sapiens; RNA-Seq
PDX/SC PDX samples were harvested by necropsy. Dead cells were removed using a dead cell removal kit (Miltenyi Biotec, cat No. 130-090-101) and mouse cells were removed by mouse cell depletion kit (Miltenyi Biotec, cat No. 130-104-694). RD cells were dissociated by 0.05% Trypsin/EDTA for single-cell suspension. For primary tumors in frozen OCT block, cells were thawed and nuclei were extracted by TST extraction buffer described in Slyper et al. 2020, Nature Medicine. Then dissociated cells from all these three types of samples were loaded with 10X Genomics GEM-RT kit for barcoded RNA extraction, cDNA amplification and library construction. 10X Genomics v2 or v3 kits were used for library construction of the samples mentioned above, with slight modification of Larry barcoding system, by added a PCR step to grab the barcoding 3'UTR together with unique barcode for individual cells. Single cells were emulsed in droplet with unique barcodes, within the droplet, Reverse-Transcription occurred. Then the cDNA from Reverse transcription were extracted by phase separation. This was followed by cDNA amplification, library construction, which is compatible with Illumina NextGeneration Sequencing kit.