GSM7843115: Vehicle, replicate 2; Mus musculus; RNA-Seq
On Day 7, freshly collected tumor sample from each mouse was minced separately, and digested with mixed enzymes in C tubes. C tubes were attached onto the sleeve of gentle MACS dissociator before running the program 37C_m_TDK_1. Digested tissues were filtered through 70 μm cell strainers. Cells were then suspended with 1 x Red Blood Cell Lysis Solution and washed twice with DPBS + 1% BSA. The collected cells were transferred into a 2ml microcentrifuge tube. Centrifuge at 300 rcf for 10min. Remove the supernatant without disrupting the cell pellet. Add an appropriate volume of 1X DPBS + 1% BSA to obtain a concentration of 700-1,200 cells/μl. Library generation was conducted as outlined by the Single Cell 3′ Reagent Kits v3.1 user guide with appropriate modifications to the PCR cycles based on the calculated cDNA concentration (as recommended by 10X Genomics). In brief, the samples were washed and re-suspended in DPBS (Corning) + 1% BSA (Sigma-B2064) and then the appropriate volume for each sample was calculated for a target capture of 8000 cells. Following cell counting, samples were adjusted to ~1000 cells/μl and loaded onto a 10x Genomics Chip G-single cell. After GEMs (Gel Bead-In-EMulsions) generation and retrieval, samples were transferred into a PCR 8-tube strip (Eppendorf) and heat-sealed. Reverse transcription was performed using a thermal cycler (Thermo Fisher). After the reverse transcription, cDNA was recovered using Recovery Agent provided by 10x followed by a Silane DynaBead cleanup (Thermo Fisher) according to user guide. Purified cDNA was amplified for 12 cycles before cleaning up using SPRIselect beads (Beckman). Finally, libraries were sequenced on the NovaSeq 6000 Illumina platform with a 150-base pair (bp) paired-end, targeting a median sequencing depth of 50,000 read pairs/cell for each sample.