title: Illumina HiSeq 4000 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). The scRNA-Seq library was prepared using the 10x V3 Single Cell Kit, according to the manufacturer's instructions. FACS-sorted cells were washed once with PBS containing 0.04% bovine serum albumin (BSA) and resuspended in PBS. 10,000 to 20,000 cells were used for GEM generation through the 10x Chromium Controller using the 10x V3 B Chip GEM generation kit. Droplet preparation was followed by reverse transcription and cell barcoding, the emulsions were resolved and cDNA was purified using Dynabeads MyOne SILANE followed by a PCR amplification (98 °C for 45 s; 13–18 cycles of 98 °C for 20 s, 67 °C for 30 s, 72 °C for 1 min; 72 °C for 1 min), with additional use of a primer for amplification of antibody derived tags (ADTs). Amplified cDNA was then used for 3′ gene expression library and ADT library construction. For gene expression library construction, amplified cDNA was fragmented and end-repaired, double-sided size-selected using SPRIselect beads. cDNA was further amplified by PCR with sample index primers (98 °C for 45 s; 14–16 cycles of 98 °C for 20 s, 54 °C for 30 s, 72 °C for 20 s; 72 °C for 1 min), and double-sided size-selected with SPRIselect beads. For ADT library construction, antibody derived transcripts were enriched after size selection aliquoting resulting in the initial cDNA amplification step, according to the CITE-Seq protocol as originally described.