|
| Status |
Public on Feb 28, 2024 |
| Title |
UBV PHH at 45hr rep3 |
| Sample type |
SRA |
| |
|
| Source name |
Primary human hepatocytes, mouse embryonic fibroblasts
|
| Organisms |
Homo sapiens; Mus musculus |
| Characteristics |
tissue: Primary human hepatocytes, mouse embryonic fibroblasts
cell line: Primary Human Hepatocytes [BioIVT donor: UBV] and J2-3T3 male mouse embryonic fibroblasts [gift of Howard Green, Harvard Medical School]
cell type: primary human hepatocyte, mouse embryonic fibroblast
genotype: WT
treatment: circadian synchronization and RNA extraction
time: 45 hours post-synchronization
|
| Treatment protocol |
To perform circadian synchronization, the cultures were placed in Non-HEPES-buffered CO2 Independent Medium (COI, Thermofisher) with 10% (v/v) fetal bovine serum (FBS, Gemini), 1% (v/v) ITS+ (insulin/ human transferrin/selenous acid and linoleic acid) premix (BD Biosciences) and 7 ng/ml glucagon (Sigma)
|
| Growth protocol |
Hepatocytes were maintained in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM with L-glutamine, Corning) with 10% (v/v) fetal bovine serum (FBS, Gemini), 1% (v/v) ITS+ (insulin/ human transferrin/selenous acid and linoleic acid) premix (BD Biosciences), 7 ng/ml glucagon (Sigma), 40 ng/ml dexamethasone (Sigma), 15 mM HEPES (Gibco), and 1% (v/v) penicillin-streptomycin (Corning). J2-3T3 male murine embryonic fibroblasts were cultured at <18 passages in fibroblast medium comprising of DMEM with high glucose, 10% (v/v) bovine serum (Thermo Fisher), and 1% (v/v) penicillin-streptomycin (Corning). Primary human hepatocytes were seeded on collagen-coated micropatterned plates as detailed previously (March et al., 2015).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using TRIzol (Thermo Fisher) and purified using RNeasy Mini Kit (Qiagen)
RNA integrity was determined using an Agilent Fragment Analyzer and purity and quantity determined using a Thermo Scientific Nanodrop1000 Spectrophotometer. High throughput RNAseq libraries were prepared as previously described (M using 100ng of total RNA input and sequenced on a NovaSeq6000 using 50nt paired-end sequencing
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Illumina NovaSeq 6000 using v3 chemistry. NovaSeq Software: Control v1.6.0, RTA 3.4.4. BCL files were converted to fastq using bcl2qseq. Indexes were split using custom scripts allowing 1 mismatch.
Gene expression was quantified using RSEM version 1.3.1 and STAR version 2.7.1a alignment to a transcriptome derived from the human hg38 primary assembly and ensembl version 98 annotation.
Assembly: hg38 with the ensembl 98 annotation
Supplementary files format and content: 190712Bha_set1_intCt.txt.gz is a tab delimited text file of integer count data
Supplementary files format and content: 190712Bha_set1_l2tpm.txt.gz is a tab delimited text file of log2 tpm data including a +1 offset
|
| |
|
| Submission date |
Nov 17, 2022 |
| Last update date |
Feb 28, 2024 |
| Contact name |
Charles Arthur Whittaker |
| E-mail(s) |
charliew@mit.edu
|
| Organization name |
Koch Institute
|
| Street address |
77 Mass Ave 76-189
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02152 |
| Country |
USA |
| |
|
| Platform ID |
GPL25526 |
| Series (2) |
| GSE218242 |
Circadian rhythm-dependent programs control inflammatory responses and drug metabolism in primary human hepatocytes I. |
| GSE218244 |
Circadian rhythm-dependent programs control inflammatory responses and drug metabolism in primary human hepatocytes |
|
| Relations |
| BioSample |
SAMN31775748 |
| SRA |
SRX18298338 |
