organism: Homo sapiens
description: Protocols: Cells were grown in appropriate media, in T75 flasks for 24 hours, media was removed and then cells were washed twice with PBS. TRIzol was added directly to the cells. Cells were split at a 90% confluent culture by digesting the monolayer in 5mL of 0.05% Trypsin-EDTA for 10 minutes or until cells lifted. The cell suspension was collected in a conical tube and centrifuged at 700xg for 7 minutes to ensure pelleting of the cells. Trypsin was removed, and cells were resuspended in media for plating, then plated at a 1:10 dilution in a new T75 flask in fresh IMDM with 5% HIFBS, 200 units/mL penicillin, and 200 µg/mL streptomycin for maintenance of line. Following addition of TRIzol, samples were transferred to 1.5 mL tubes. Preparation of RNA was done following manufacturers instructions. Approximately 500 ng of protein-free and intact total RNA was used for library construction using the reagents provided in the NEBNext Ultra II RNA Library Prep following the manufacturer's protocol. First, 2 µL of diluted RNA was spiked with ERCC from the kit. Next, mRNA isolation was performed using the NEBNext Poly(A) mRNA Magnetic Isolation module (New England Biolabs). Then RNA was fragmented in a solution containing divalent cations, with incubation at 94°C. Next, first strand cDNA synthesis using reverse transcriptase and random primers was done. Synthesis of double stranded cDNA was done using the second strand master mix provided in the kit, followed by end-repair and dA-tailing. At this point, Illumina adaptors were ligated to the sample. Finally, the library was enriched by 11 cycles of amplification and purified by Agencourt AMPure beads (Beckman Coulter). Barcoded libraries were sized on the bioanalyzer and quantitated by QUBIT. Quantitative PCR was used to validate the library's functionality, using the KAPA Library Quantification kit (Kapa Biosystem) and monitoring with the BioRad Touch Real-Time PCR Detection System. Individual libraries were pooled equimolarly for sequencing runs.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2018-09-01T17:02:14Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-06-19T11:37:49Z
attributes.tag: External Id
attributes.value: SAMEA4743760
attributes.tag: INSDC center name
attributes.value: UNIVERSITY OF FLORIDA
attributes.tag: INSDC first public
attributes.value: 2018-09-01T17:02:14Z
attributes.tag: INSDC last update
attributes.value: 2018-06-19T11:37:49Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6919:HC-04 I1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: HC-04
attributes.tag: cell type
attributes.value: hepatocyte
attributes.tag: common name
attributes.value: human
attributes.tag: disease
attributes.value: hepatoblastoma
attributes.tag: growth condition
attributes.value: IMDM
attributes.tag: organism part
attributes.value: liver
attributes.tag: phenotype
attributes.value: wild type
attributes.tag: replicate
attributes.value: B01
attributes.tag: sample name
attributes.value: E-MTAB-6919:HC-04 I1
attributes.tag: scientific_name
attributes.value: Homo sapiens