|
| Status |
Public on Feb 28, 2024 |
| Title |
BMAL knock-down 36h rep 2 |
| 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
treatment: BMAL siRNA
time: 36 hour post-synchronization
|
| Treatment protocol |
siRNA oligonucleotides were added to patterned hepatocytes after washing off the unbound hepatocytes. ON-TARGETplus SMARTpool siRNA oligonucleotides (Dharmacon) were delivered to hepatocytes by using RNAiMAX Transfection Reagent (Thermo Fisher Scientific) per manufacture’s protocols at final concentration 100nM in antibiotic-free DMEM media supplemented with 10% FBS (final volume 100 mL). Duplicate or triplicate wells-containing hepatocytes were exposed to siRNA duplexes overnight for 20-24 hr and subsequently surrounded by 3T3-J2 fibroblasts. 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). 36, 42, 48 and 54 hour post-synchronization, RNA was extracted.
|
| 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 v1.7.0, 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 Salmon version 1.3.0 with a transcriptome target consisting of human and mouse transcripts. Human transcripts were derived from the hg38 primary assembly and the ensembl version 101 annotation and mouse transcripts were derived from the mm10 primary assembly and the ensembl 101 annotation. Gene level summaries were prepared using tximport version 1.18.0 running under R version 4.0.3.
Assembly: hg38 and mm10 with ensembl version 101 assembly
Supplementary files format and content: 210308Bha_set3_intCt.txt.gz is a tab delimited text file of integer count data
Supplementary files format and content: 210308Bha_set3_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) |
| GSE218243 |
Circadian rhythm-dependent programs control inflammatory responses and drug metabolism in primary human hepatocytes II. |
| GSE218244 |
Circadian rhythm-dependent programs control inflammatory responses and drug metabolism in primary human hepatocytes |
|
| Relations |
| BioSample |
SAMN31775719 |
| SRA |
SRX18298268 |
