NextSeq 500 sequencing; Regional differences in human biliary tissues and corresponding in vitro derived organoids
Regional differences in human biliary tissues and corresponding in vitro derived organoids
Samples included: (1) epithelial enriched primary tissue samples from human extrahepatic Gallbladder (GBD), Common Bile Duct (CBD) and Pancreatic Duct (PancD) tissues isolated as described below, (2) Extrahepatic bile duct organoids derived from these epithelial tissue samples and cultured for 5 passages as described below and (3) Intrahepatic bile duct organoids derived from human liver tissue cultured in Extrahepatic media conditions (described below) or alternatively in media conditions as described in the publication in Cell by Huch et al (2015). GBD samples were received as either whole GBDs or small 2 cm2 tissue segments. Whole GBDs were drained of bile and the neck discarded. A longitudinal incision was used to expose the GBD mucosal surface. CBD and PancD samples were received intact and the wall of the ducts incised with a scalpel to expose the lumen. Tissues were washed three times with cold Hanks Balanced Salt Solution (HBSS). Tissues were transferred to cold ADV/DMEM F12 with 100 U/mL penicillin and 100 μg/mL streptomycin. The mucosal surface was then abraded with a scalpel to mechanically dissociate the epithelium (Figure 1A). Cells were centrifuged at 300 x g for 5 min, washed twice, then re-suspended in growth factor reduced (GFR) matrigel. 50 μL droplets were plated per well into a 24-well plate. Matrigel was allowed to solidify for 5-10 min at 37°C before adding 500 μL of ADV/DMEM F12 containing 1x N2-serum free supplement, 1x B27-serum free supplement, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. The following growth factors were added: 20% R-spondin conditioned media or 500 ng/mL recombinant human R-spondin 1, 3 μM CHIR 99021, 100 ng/mL recombinant human noggin, 2.5 μM PGE2, 100 ng/mL recombinant human EGF, 5 μM A 83-01, and 10 μM forskolin (FSK). For the first 48 h, 10 μM Y-27632 was added to the media. Media was changed every 2-3 days. Total RNA was extracted using either the RNeasy Mini or Micro Kit (Qiagen) depending on the expected RNA yield. DNAse treatment was performed for all samples. TruSeq Stranded mRNA Library Prep Kit for NeoPrep (Catalog # NP-202-1001) was used for library construction using 100 ng Total RNA input, with default settings of 200 bp target insert size and 15 cycles PCR (Protocol Version 1.1.7.6).