GSM3267827: wt_txm; Mus musculus; RNA-Seq
For each dataset, cell lines were mixed at equal concentrations and the resulting pool was prepared for 10X Chromium scRNA-seq 48h later. Cells were single-cell dissociated with Accutase (Sigma-Aldrich), centrifuged and resuspended in 1mL pre-chilled DPBS+0.04%BSA. Cells were counted and resuspended in DPBS+0.04%BSA following the 10xChromium Volume Calculator Table for a total of 10,000 targeted cells Preparation of 10X Chromium scRNA-seq transcriptome libraries for sequencing was carried out largely using the standard protocol. In order to maintain gRNAs in the library, which are shorter than standard transcripts, SPRI purification was altered as follows: purification of cDNA was carried out with 1.6X beads instead of the standard 0.8X, post-fragmentation purification was performed with 1.6X instead of 0.8X, post-adapter ligation purification was performed with 1.4X instead of 0.8X, and post-SI PCR was performed with 1.2X instead of 0.8X. Additionally, to reserve a portion of cDNA and allow for custom applications such as gRNA sequence capture, two additional PCR cycles (12 instead of 10) were performed at the cDNA amplification stage. Half of the product was used for the transcriptome prep, ¼ was amplified to obtain the required amount for gRNA sequence capture, and 1/8 was used for gene-specific PCR enrichment.
GEO Accession
GSM3267827