NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM6737796 Query DataSets for GSM6737796
Status Public on Feb 28, 2024
Title BMAL knock-down 36h rep 1
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 SAMN31775720
SRA SRX18298267

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap