GSM6701493: E9.5, replicate 1, scRNAseq; Mus musculus; RNA-Seq
Embryos were harvested at E9.5, E12.5, E15.5, and embryos from the same stages were pooled together. The tail, limbs, and liver were removed from E12.5 and E15.5 embryos. The rest tissue or whole E9.5 embryos were dissected into small pieces using sterile scalpels and washed three times with Hank's Balanced Salt Solution (HBSS) to remove most blood cells, then were incubated in 2 mg/ml Collagenase Type II (Worthington, Cat# LS004174) and 50% (v/v) Accutase (StemCell Technologies, Cat# 07920) solution for 15 mins at 37℃. The sample was further treated with gentleMACS Dissociator (Miltenyi Biotec, Germany) twice to get single-cell suspension. The enzyme was neutralized by 2% FBS -HBSS. The cell suspension was filtered through a 40-µm cell strainer (Corning). After centrifugation (300g, 5 min, 4 °C) the supernatant was removed and the pellet was resuspended in flow buffer (1× phosphate-buffered saline buffer [PBS], 0.5% bovine serum albumin) and then incubated with Rat anti-human/mouse CD31-FITC (Thermo Fisher, Cat# 11-0311-82) for 15 mins at room temperature. Then Magnetic-Activated Cell Sorting (MACS) was performed with EasySep™ FITC Positive Selection Kit II (StemCell Technologies, Cat# 17682) to enrich ECs. The purified cells were resuspended in DMEM + 10% FBS. The viability and cell number of enriched cellular samples were determined by Countess Automated Cell Counter (Thermo Fisher). Library was performed according to the manufacter's instructions (single cell 3' v1 or v2 protocol, 10x Genomics). Briefly, ECs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the A chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.