GSM3557469: P09_Ctrl20-37576824_single_cell_passage_09; Homo sapiens; RNA-Seq
Frozen cells from different passages were thawed and plated in T75 flasks for 5-7 days. Once they reached 70-80% confluence, cells were collected for single cell separation. Fluidigm C1 Single-Cell Auto Prep Array for mRNA-Seq (10-17um) and Fluidigm C1 system were used for cell capture following the Fluidigm protocol, then reverse transcription and cDNA amplification were performed using the SMARTer PCR cDNA Synthesis kit (Clontech, version 1) on the C1 unit. Single-cell cDNA from validation cells were harvested for library preparation. Nextera XT DNA sample preparation kits and Index kits were used for library preparation following the manufacturer's protocol. Single-cell cDNA libraries from each passage were pooled and cleaned up, then cDNA size distribution was examined by 2100 Bioanalyzer. This was followed by quantification and normalization with the KAPA Library Quantification Kit and BioRad CFX96 qPCR machine (BioRad). Single cell mRNA sequencing was performed on NextSeq 500 by using the NextSeq High Output v2 kit (300 cycles). PhiX Control v3 (Illumina) was used as a quality control for sequencing runs.
GEO Accession
GSM3557469