GSM8323313: Day3_DB-11-BE87_replicate2; Mus musculus; RNA-Seq
At designated times (day 0, day 1, day 3, day 11) cells were removed from Matrigel. Briefly, media was removed and washed with ice cold PBS. TrypLE Express (Gibco; 12604013) was added to wells and incubated at 37°C, followed by scraping of Matrigel bubbles to collect in a 15 mL conical tube. Cells were mechanically disrupted by pipetting ~10 times and incubated at 37°C for an additional 10 minutes. Cells were mechanically disrupted again and enzyme was inactivated with DMEM/F12 + 10% FBS + 1/100 Penicillin-Streptomycin (Gibco; 15140122). Cells were centrifuged at 300g at 4°C, resuspended to 1000 cells/ μL in DMEM/F12 + 10% FBS, and filtered through a Flowmi 40 μM cell strainer (Sigma; BAH136800040). The Chromium Next GEM Single Cell 3' reagent kit v3.1 from 10X Genomics (protocol CG000204 Rev C) was used in scRNA-seq library preparation. Single-cells were partitioned into nanoliter-scale Gel Beads-inemulsion (GEMs) using Chromium Controller. Upon cell lysis, the poly-A tails of mRNA are captured on the beads containing an Illumina read 1 sequencing primer, 16nt 10X barcode, 12nt UMI and 30nt poly (dT) sequence. Reverse transcription occurs followed by amplification of full-length cDNA from polyA mRNA. Enzymatic fragmentation and size-selection was used to optimize cDNA amplicon size. End repair, Atailing, Adaptor ligation and PCR create the final sequencing libraries that contain the P5 and P7 primers used in Illumina bridge amplification. The libraries were sequenced using NextSeq 550 High Output Kit v2.5 (150 cycles).