title: CBD Organoids 6_TechRep4
organism: Homo sapiens
description: Protocols: 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).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-01-20T17:03:18Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-01-14T09:29:06Z
attributes.tag: External Id
attributes.value: SAMEA5216234
attributes.tag: INSDC center name
attributes.value: Wellcome MRC Cambridge Stem Cell Institute University of Cambridge
attributes.tag: INSDC first public
attributes.value: 2019-01-20T17:03:18Z
attributes.tag: INSDC last update
attributes.value: 2019-01-14T09:29:06Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7569:CBD Organoids 6_TechRep4
attributes.tag: age
attributes.value: 48
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: growth condition
attributes.value: organoid culture (extrahepatic medium)
attributes.tag: individual
attributes.value: 22
attributes.tag: organism part
attributes.value: common bile duct
attributes.tag: sample name
attributes.value: E-MTAB-7569:CBD Organoids 6_TechRep4
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: sex
attributes.value: female
attributes.tag: study group
attributes.value: CBD Organoids