Transcriptomes of adult lymphatic endothelial cells from wildtype and Foxc2lecKO mice

Single cell RNA sequencing of mesenteric lymphatic endothelial cells from adult willdtype and Foxc2lecKO mice Methods: single cell RNAseq profiles of lymphatic endothelial cells sorted from WT and Foxc2lecKO were generated by 10x sequencing, in triplicate. The sequencing libraries prepared following the manufacturer’s recommendations (Chromium Single Cell 3ʹ GEM Kit v3). Briefly, an emulsion encapsulating single cells, reverse transcription reagents and cell barcoding oligonucleotides was generated. After the actual reverse transcription step, the emulsion was broken and double stranded cDNA generated and amplified in a bulk reaction for 12 PCR cycles. This cDNA was fragmented, a P7 sequencing adaptor ligated, and a 3’ gene expression library generated by a 14 cycles PCR amplification. Libraries were quantified by a fluorimetric method (QubIT, Life Technologies) and their quality assessed on a Fragment Analyzer (Agilent Technologies). Cluster generation was performed with 1.4nM of an equimolar pool from the resulting libraries, including 10% PhiX spike, using the Illumina HiSeq 3000/4000 PE Cluster Kit reagents (Illumina). Sequencing was performed on 2 runs in the Illumina HiSeq 4000 using HiSeq 3000/4000 SBS Kit reagents (Illumina) according to Illumina 10X Genomics recommendations. Sequencing data were demultiplexed using the bcl2fastq2 Conversion Software (version 2.20, Illumina) and primary data analysis performed with the Cell Ranger Gene Expression pipeline (version 3.1.0, 10X Genomics). A minimum of 33.000 reads/ cell were obtained and 18.271 genes on average were detected.