title: Illumina NovaSeq 6000 paired end sequencing; Immune cells direct epithelial maturation during early human lung development
design: Immune cells direct epithelial maturation during early human lung development
library_name: FL9_p
library_construction_protocol: Lung tissue for this project was obtained from the MRC-Wellcome Trust Human Developmental Biology Resource (https://www.hdbr.org). The HDBR obtain human embryonic and fetal samples from medical or surgical terminations of pregnancy. We requested lung tissue samples from fetuses aged between 8 and 20 post-conception weeks (pcw). We received whole lungs or lung fragments in refrigerated Hibernate E medium. Any trachea or non-lung tissue was removed by dissection. To enrich lung samples for CD45+ immune cells, we utilised fluorescence activated cell sorting (FACS). Cells were first blocked, to prevent non-specific antibody binding, using Human TruStain FcX (Biolegend, 422301) and then stained for 30 minutes on ice, using flurochrome-conjugated antibodies, CD45-PE, CD31-BV421, EpCAM-APC and CD235a-FITC. After staining, cells were resuspended in buffer containing DAPI, to enable the determination of live versus dead cells. FACS was performed on a BD FACS Aria. Appropriate 'fluorescence minus one' controls were used to determine FACS gating. Samples were gated to allow the collection of live single cells that were negative for CD235a (red blood cells), CD31 (endothelial cells) and EpCAM (epithelial cells), and positive for CD45 (immune cells). CD45+ immune cells were frozen prior to CITE-Seq or scRNAseq. Nucleic acid was extracted according to the standard protocol of the Chromium single cell 5'V1 kit. CD45+ cells were thawed and counted, assessing viability using Trypan Blue. Cells were stained using the TotalSeq™- C antibody panel (Biolegend) where possible (some samples were not stained), prior to immediate processing for 10x 5' single-cell capture using the Chromium Next GEM Single Cell V(D)J Reagent Kit, v 1.1, according to the manufacturer's protocol. The Chromium Next GEM Single Cell V(D)J Reagent Kit, v 1.1 was used for scRNAseq library construction for unstained samples. The same Kit with Feature Barcoding Technology for cell surface proteins was used for those samples stained with antibodies.