Ocug 1

The GBC cell lines, OCUG-1 and NOZ were obtained from Health Science Research Resources Bank, Osaka, Japan. TGBC2TKB, TGBC24TKB and G-415 were purchased from RIKEN Bio Resource Center, Ibaraki, Japan. SNU-308 was obtained from Korean Cell Line Bank, Seoul, Korea. GB-d1 was authenticated by short tandem repeat analysis. The properties and culture conditions of the GBC cell lines, TGBC2TKB, SNU-308, G-415, TGBC24TKB, NOZ, OCUG-1 and GB-d1 are provided in Additional file 1. All cell lines were maintained in humidified incubator with 5 % CO₂ at 37 °C.

The GBC lines used in this study were as follows: GBC-SD, SGC-996 were purchased from Shanghai Institute for Biological Science, Chinese Academy of Science (Shanghai, China). NOZ, OCUG-1, EHGB-1, EHGB-2 were purchased from the Health Science Research Resources Bank (Osaka, Japan). GBC-SD, EHGB-1, EHGB-2, OCUG-1 cells were cultured separately in DMEM (Gibco) with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin–streptomycin (Gibco). NOZ cells were cultured in William’s medium E (Lonza) with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin–streptomycin (Gibco). Contrastingly, SGC-996 cells were cultured in RPMI 1640 medium (Hyclone) with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin–streptomycin (Gibco). All of above cells were cultured in their respective media in a humidified incubator at 37 °C with 5% CO₂.

In this study, we analyzed 167 human primary GBC samples as well as 39 non-GBC samples and the corresponding matched normal tissue in most cases using exome-seq, and/or low-pass whole-genome sequencing and/or RNA-seq (Supplementary Table 1). Fresh frozen samples used in the study were obtained from patients undergoing extirpative surgery for GBC. This study was conducted with IRB approval (Pontificia Universidad Católica de Chile IRB, Institutional Human Ethics Committee of Jiwaji University (India) and Seoul National University Hospital IRB (Seoul)) and written patient informed consent. Human tissue samples were de-identified prior to their shipment and analysis and are not considered human subject research under the US Department of Human and Health Services regulations and related guidance (45 CFR Part 46). Basic demographic information for the patient samples in the study, where available, is included in Supplementary Data 1. Tissue processing as well as simultaneous extraction of high-quality genomic DNA and total RNA from the same samples were performed as previously described^{47 (link)}. The study also included GBC cell lines TGBC24TKB, TGBC2TKB, G-415 (RIKEN Bio Resource Center, Ibaraki, Japan), OCUG-1 (Health Science Research Resources Bank, Osaka, Japan), SNU-308 (Korean Cell Line Bank, Seoul, Korea), GB-d1 (From Dr. Masao Tanaka’s lab, Japan)^{48 (link)}.

The human GBC cell lines NOZ and OCUG-1 were purchased from the Health Science Research Resources Bank (Osaka, Japan). The cells were cultured in Dulbecco’s modified Eagle’s medium with L-glutamine (Wako Pure Chemical Industries, Ltd., Osaka, Japan) supplemented with 1% penicillin/ streptomycin (Gibco BRL, Grand Island, NY, USA) and 10% fetal bovine serum (Gibco BRL). The cells were incubated with 5% carbon dioxide at 37°C.

Cell lines used in the current study were grown as per vendor recommendations. Briefly the cells were cultured in their respective media as detailed in Additional file 1: Method S1 and 1% penicillin/streptomycin mixture at 37 °C in a humidified 5% CO₂ atmosphere. Cells were harvested at 70% confluency. Gall bladder cancer cell lines G-415 was sourced from RIKEN Bio Resource Center, Ibaraki, Japan and OCUG-1 and NOZ from Health Science Research Resources Bank, Osaka, Japan. Bladder cancer cell lines were received from Prof. Jean Paul Thiery (Department of Biochemistry, National University of Singapore, Singapore).