GSM9032275: 3 pooled E9.5 microdissected Tbx1Cre/+; Kmt2d F/+; GFP F/+ Embryos; Mus musculus; RNA-Seq
Embryos were collected at E9.5 and microdissected in 1x PBS at 4 °C. Dissected tissue was maintained in DMEM (Dulbecco's Modified Eagle Medium, GIBCO 11885084) at 4°C while genotyping was performed. Microdissections included the pharyngeal region from PA1 onwards and included the heart. Embryos were pooled by genotype and then incubated in 1 ml of 0.25% Trypsin-EDTA (GIBCO, 25200056) containing 50 U/ml of DNase I (Millipore, 260913-10MU), at room temperature for 7 min with occasional pipetting to break up the tissue. 100 uL of FBS (fetal bovine serum, ATCC, 30-2021) kept at 4°C was added to stop the reaction at a final concentration of 10%. Dissociated cells were centrifugated for 5 min at 300 × g at 4 °C and the supernatant was removed. Cells were then resuspended in 1x PBS (without Ca2+ and Mg2+, Coming, 21-031-cv) containing 10% FBS at 4 °C. These preparations were then filtered with a 100-μm cell strainer and 1 μl DAPI (1 mM) (Thermo Fisher Scientific, D3571) was added to each sample. Cells were FACS (Fluorescence Activated Cell Sorting) purified using a BD FACSAria II (BD Biosciences) system and BD FACSDiva 8.0.1 software in order to collect EGFP-positive and DAPI negative living cells in 1x PBS containing 10% FBS at 4°C. Following collection, these cells were centrifuged at 4 °C for 5 min at 300 × g. Finally, supernatant was removed and the cells were resuspended in 50 μl of 10% FBS. Library preparation was performed using standard manufacter's instructions using the 10X Genomic Chromium GEM-X Single Cell 3ʹ Kit v4.1 PN-1000691.  Libraries were indexed using the Dual Index Kit TT Set A  PN-1000215 and standard Illumina adaptors. The sequencing primers are standard Illumina, i5 and i7.
GEO
GSM9032275_r1