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Sample GSM7869864 Query DataSets for GSM7869864
Status Public on Apr 20, 2024
Title HDFs - Isolated FA S2
Sample type SRA
 
Source name Human Dermal Fibroblasts
Organism Homo sapiens
Characteristics cell line: Human Dermal Fibroblasts
cell type: Fibroblasts
genotype: WT
sample type: Isolated Focal Adhesions
Treatment protocol Both whole cell and isolated FA samples were seeded down on fibronectin. Isolated FA samples were rinsed once with PBS and then treated for 3 minutes in a 2.5 mM triethanolamine (Sigma, 90279-100ML) low ionic strength buffer, pH 7.0. Immediately after the incubation, cell bodies were removed using hydrodynamic force (setting 1, Interplak, Conair) using PBS at ~0.5 cm from and ~90° to the surface of the dish. Isolated FA or whole cell samples were collected in trizol for RNA Extraction
Growth protocol HUVECs were maintained on on dishes coated with 0.1% w/v gelatin (10 min at room temperature in PBS; Sigma) in M199 medium (11150-059, Thermo Fisher) supplemented with 20% FBS (F0926-500 ML, Sigma), supplemented with 10 µg/mL endothelial cell growth supplement prepared from bovine hypothalamus, 50 µg/mL heparin filtered (H3149-100KU, Sigma), and 1% Penicillin-Streptomycin (15140122, Thermo Fisher). HDFs were cultured in fibroblast growth medium (Fibroblast Growth Kit-Low Serum; ATCC, PCS-201–041) supplemented with 0.5 mL Phenol Red (ATCC, PCS-999-001, final concentration 33 µM) and 1% Penicillin-Streptomycin (ThermoFisher, 15140122).
Extracted molecule polyA RNA
Extraction protocol Trizol was used to extract RNA.The quality was verified using the 2100 Bioanalyzer.
500 ng of total RNA was used to prepare Lexogen QuantSeq 3′ mRNA-Seq FWD libraries for Illumina deep-sequencing according to the manufacturer’s protocols. Libraries were amplified for 13-14 PCR cycles.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
 
Description HDF_adhesion_B2
Data processing Raw reads were processed using LabxPipe (https://github.com/vejnar/LabxPipe): first reads were trimmed using ReadKnead (https://github.com/vejnar/ReadKnead) using the “bktrim” algorithm using the sequence AAAAAAAAAAAAAAAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC. They were then mapped to the human genome using STAR with non-default parameters “–alignEndsType Local” and “–seedSearchStartLmaxOverLread 0.8”. FON1 files containing genes (or “metagenes”) were generated by concatenating the isoforms of each gene together using the “–method union” option of the ”fon_transform” tool. Read counts per gene were computed by summing the total number of reads overlapping at least 10 nucleotides of the gene (i.e. metagene) annotation. Reads mapping to multiple loci were accounted for by dividing 1 (each read) by the number of loci to which the read was mapped to. To identify differentially expressed genes, genes with at least 1 count in both conditions in any of the replicates were first selected, and input to the “DESeq” function. The “results” function with parameters “pAdjustMethod=”fdr”, independentFiltering=FALSE” returned the differentially expressed genes.
Assembly: GRCh38
Supplementary files format and content: comma separated values file with raw counts for each sample
 
Submission date Oct 29, 2023
Last update date Apr 20, 2024
Contact name Liana Boraas
E-mail(s) liana.boraas@yale.edu
Phone 2037376480
Organization name Yale University
Street address 300 George Street, Room 752: Loria Center
City New Haven
State/province CT
ZIP/Postal code 06511
Country USA
 
Platform ID GPL16791
Series (2)
GSE246499 Translationally inhibited mRNAs control cell movement as untranslated sequences [RNA-seq]
GSE246956 Translationally inhibited mRNAs control cell movement as untranslated sequences
Relations
BioSample SAMN38030644
SRA SRX22258983

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA

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