title: Illumina NovaSeq 6000 paired end sequencing; Single-cell Atlas of intrahepatic T cells reveals a population of liver-resident naive-like CD4+ T cells expanded in primary sclerosing cholangitis
design: Single-cell Atlas of intrahepatic T cells reveals a population of liver-resident naive-like CD4+ T cells expanded in primary sclerosing cholangitis
library_construction_protocol: We conducted a single-centre cross-sectional study in adult patients (>18 years of age) undergoing liver transplantation (LTX) for PSC (n=19), ALD (n=16) or resection for PSC-associated cholangiocarcinoma (to which we refer as PSC early-stage due to absence of cirrhosis n=2) at the University Medical Center Hamburg-Eppendorf (UKE, Hamburg, Germany). Liver tissue and whole blood were obtained from each patient at the time of surgery. Diseases were diagnosed and treated following the guidelines of the European Association for the Study of the Liver (EASL). Clinical parameters were recorded immediately before surgery. PSC patients with earlier disease stages (PSC early, n=8) were recruited from our outpatient clinic for phenotyping and functional experiments from peripheral blood. Sex and age matched healthy blood donors (HD, n=10) were used as additional controls.  All patients and donors participating in this study provided written informed consent according to the ethical guidelines of the Institutional Review Board of the medical faculty of the University of Hamburg (PV4081 & PV4898). scATAC-Seq libraries were prepared using the 10x genomics single-cell NextGEM ATAC-Seq Kit v1.1 according to manufacturerâ€™s instructions. FACS sorted naive CD4+ T cells from blood of PSC patients and healthy donors were centrifuged, cell pellets were resuspended in PBS containing 0.04% bovine serum albumin (BSA). Nuclei isolation was performed by centrifugation, cell lysis and resuspension according to the demonstrated protocol for nuclei isolation (CG000169). Nuclei were counted and processes according to the low input protocol (<40.0000 nuclei). GEM generation through the 10x Chromium Controller using the 10x V1.1 H NextGEM Chip. Droplet preparation was followed by barcoding and transposition. Emulsions were resolved and single-stranded DNA was purified using Dynabeads MyOne SILANE followed by library construction and sequencing.