GSM8988416: Mouse 31, Sample 31-1 (LTT1a), Lower trunk and tail, E7.5 tdTomato-virus (pMR671), GEX; Mus musculus; RNA-Seq
The embryos were removed from the uterus, sacrificed, and placed into a petri dish containing Hank's Balanced Salt Solution (HBSS) on ice. Embryos were screened for expression of Tomato or GFP using a benchtop fluorescence stereoscope. Limbs, liver, and the ventral skin were removed. Body regions, primarily lower trunk, middle trunk, and cranial regions, were dissected separately and placed into a small Petri dish containing 2.5 mL of a cell dissociation buffer consisting of 2mg/mL Collagenase P (Roche, Sigma-Aldrich), 1X TrypLE, and 1uL/mL DNASe I, fully dissolved in HBSS (without phenol red, including Ca2+ and Mg2+). Tissue was chopped finely with a scalpel and forceps under control of stereomicroscope and transferred to 15mL falcon tubes. The tissue pieces were triturated 10X using a transfer pipette, and then 10X using a 1mL pipette. Samples were then incubated for 20 minutes at 37 C in a preheated shaking incubator set at 150 RPM, with the tubes tilted to ~60 degrees. During this time every 6-8 minutes, the samples were vortexed and triturated using a 1mL pipette. After the tissue clumps disappeared, 12.5 mL of 2% Fetal Bovine Serum (FBS) diluted in ice-cold HBSS (without phenol red, without Ca2+ and Mg2+, H6648 SIGMA) was added. The samples were centrifuged in a cold centrifuge (4C) for 15 minutes at 500 g. The supernatant was carefully removed and the pellet was resuspended in 1mL HBSS + 2% FBS and kept on ice thereafter. After centrifugation and resuspension, the cells were filtered into round-bottom polypropylene test tubes (Falcon) using a cell strainer with a 40 micrometer woven polyethylene terephthalate mesh (pluriSelect pluriStrainer SKU 43-10040-40) for downstream cell sorting. Cells were loaded into the Chromium machine (aiming for ~10,000 cells per reaction) using the V3.1 single-cell reagent kit (10X Genomics). Following GEM creation and cell lysis, cDNA was synthesized by reverse transcription, then amplified by PCR for 12 cycles as per the manufacturer's protocol (10X Genomics). A portion of the amplified cDNA was used to generate Gene expression libraries and then indexed using the Single Index Plate T Set A. CloneID enrichment from cDNA libraries was additionally performed based on Ratz et al 2022 to increase the retrieval of the barcodes by ~5-10%. Briefly, two nested PCR reactions were performed using Q5 error-correcting DNA polymerase to amplify the CloneID-containing region, and a third reaction was used for sample indexing using the Single Index Plate T Set A. In between each reaction, amplicons were purified by 0.8X AMPure beads using a DynaMag Magnetic separator. Indexed GE and CloneID-enrichment libraries were quantified using BioAnalyzer and/or Qubit, and then pooled prior to Illumina sequencing on a NovaSeq 6000 S2-200 v1.5 flow cell with read setup 28-8-0-166.