title: Illumina HiSeq 2000 paired end sequencing; Single-cell RNA sequencing of VE-Cadherin+ cells isolated from mouse E10.5 aorta-gonad-mesonephros region.
design: Single-cell RNA sequencing of VE-Cadherin+ cells isolated from mouse E10.5 aorta-gonad-mesonephros region.
library_construction_protocol: Embryos from pregnant mice were collected at 10.5 days of pregnancy. A total of 15 embryos was dissected to isolate the aorta-gonad-mesonephros (AGM) region. The fifteen AGM samples were combined and stained with antibodies specific for VE-Cadherin (endothelial specific) and CD41(blood specific) cell surface markers. Cells expressing VE-Cadherin were sorted on a FACS sorter and loaded on a Fluidigm C1 10–17 uM IFC Chip. Annotation of cell capture was carried out on a brightfield microscope at 40x magnification and each captured site was noted for having single-cell capture, doublet cell capture or debris. A total of 83 chambers out of the 96 in the IFC chip contained single cells. Lysis, reverse transcription, and PCR reagents (Clontech Takara) were then loaded into appropriate inlets as per Fluidigm layout. ERCC spike-in controls (Ambion) were added at a dilution of 1:4000 within the lysis mix to gain limit of detection and normalisation in downstream analysis. Illumina library preparation was carried out using the Nextera XT DNA system (Illumina), but volumes were reduced as per the modified Fluidigm C1 protocol. Tagmentation was performed on all 96 samples using 1.25 ul of cDNA (125 pg total concentration) combined with 2.5 ul of tagmentation buffer (TD) and 1.25 ul of Amplicon Tagment Mix (ATM), and incubated for 10 min at 55 degrees. After incubation, 1.25 ul of Neutrilize Tagment Mix (NT) was added. Illumina PCR barcoded indexes were combined to obtain 96 distinct combinations of i7 and i5 barcodes, and 2.5 ul of combined index was added along with 3.75 ul of Nextera PCR Mix (NPM). Samples were then put through 12 cycles of PCR as per the Illumina protocol. Each column was then pooled into a 0.2 mL 8-strip PCR tube and Ampure XP purification was performed, after which each pool was combined to obtain a single 96-sample multiplexed pool of barcoded library. The final sequencing library was quantified by Qubit (Life Technologies) and size distribution was measured on an Agilent BioAnalyzer.
attributes.tag: Experimental Factor: single cell identifier
attributes.value: cell 67