alias: E-MTAB-8303:3D_hic_p
title: HiSeq X Ten paired end sequencing; Epithelial cells in 2D and 3D culture exhibit significant differences in long-range genomic interactions
design: Epithelial cells in 2D and 3D culture exhibit significant differences in long-range genomic interactions
library_construction_protocol: The cell were collected for the Hi-C experiments or extract RNA directly. AML12 (alpha mouse liver 12) cells were cultured in a mix of Dulbecco`s modified Eagle's medium and Ham's F12 medium (1:1) containing 10% fetal calf serum and supplied with 1× ITS liquid media supplement (10 μg/mL recombinant human insulin, 5.5 μg/mL human transferrin (substantially iron-free) and 5 ng/mL sodium selenite, Sigma), dexamethasone (40 ng/mL) (Sigma), Penicillin-Streptomycin (100 U/mL) (Gibco) at 37°C for 48 h. Then, the cells were dissociated with 0.25% trypsin to suspend for replating. For the 2D cultures, cells were replated in 10 cm dishes. For the 3D cultures, cells were grown in Matrigel-embedded 3D cultures in the 10 cm dish. When the cells in the 2D cultures were 50% confluent and the cells in the 3D cultures had grown for 24 h, 1 mM hydroxyurea was added to the medium for 24 h (2D) or 36 h (3D) to arrest the cells in G1/S boundary. The Hi-C chimeric DNA were prepared by \"in-situ Hi-C\" method with minor modifications, Briefly, nuclei released from ten million crosslinked cells were digested with Mbo I (NEB). After biotin fill-in and ligation in nuclei, biotin labeled ligation product were fragmented with Cavrios M220, the molecular with size ranging from 300bp to 500bp were selected for the generation of library.Total RNA were extracted with Trizol reagent. The Hi-C libraries were prepared using NEBNext Ultra DNA Library Prep Kit for Illumina with modifications. Briefly, after end-repair and adaptor ligation with NEB modules, biotin labeled validated chimeric DNA were selected with Streptavidin C1 beads (Invitrogen). Hi-C library was amplified after DNA elution by incubating at 98°C. RNA-seq libraries were prepared using KAPA Stranded mRNA-seq kit following the manufacturer's instructions.
identifiers.namespace: Shanghai Jiao Tong university, Shanghai, China
identifiers.id: E-MTAB-8303:3D_hic_p
attributes.tag: Experimental Factor: growth condition
attributes.value: 3D culture