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Sample GSM8279792 Query DataSets for GSM8279792
Status Public on Jun 26, 2024
Title A375, Illumina, sRNA-seq, rep2
Sample type SRA
 
Source name A375
Organism Homo sapiens
Characteristics cell line: A375
Growth protocol A375 and BT474 cells were grown at 37°C with 5% CO2 in DMEM (Cellgro) supplemented with 10% FBS (Gibco), and 50 U Penicillin and 50 μg Streptomycin per mL (Gibco). Cattle and bison samples were obtained from experimental animals housed at Washington State University, Pullman, WA and University of Wyoming, Laramie WY, respectively. 2.8 ml blood was collected from healthy animals using PAXgene Blood RNA tubes (BD Bioscience) by venipuncture (jugular) and transported on ice to the laboratory for processing.
Extracted molecule total RNA
Extraction protocol Cells were washed once with ice cold DPBS (Gibco), rested on ice for 5 minutes, washed one more time with ice cold DPBS and then lysed in 1 ml TROZOL. RNA isolated as described by the manufacturer.
csRNA-seq was performed as described in (S. H. Duttke et al. 2019). Small RNAs of ~20-60 nt were size selected from 0.4-3 µg of total RNA by denaturing gel electrophoresis. A 10% input sample was taken aside and the remainder enriched for 5'-capped RNAs. Monophosphorylated RNAs were selectively degraded by 1 hour incubation with Terminator 5'-Phosphate-Dependent Exonuclease (Lucigen). Subsequently, RNAs were 5'dephosphorylated through 90 minutes incubation in total with thermostable QuickCIP (NEB) in which the samples were briefly heated to 75°C and quickly chilled on ice at the 60 minutes mark. Input (sRNA) and csRNA-seq libraries were prepared as described in (Hetzel et al. 2016) using RppH (NEB) and the NEBNext Small RNA Library Prep kit, amplified for 11-14 cycles.
 
Library strategy ncRNA-Seq
Library source transcriptomic
Library selection size fractionation
Instrument model Illumina NovaSeq 6000
 
Description short RNAs (20-65nt)
sRNA_Homo_sapiens-A375_Illumina.reps.merged.tss.txt
Data processing Reads were trimmed of their adapter sequences using HOMER (homerTools trim -3 AGATCGGAAGAGCACACGTCT -mis 2 -minMatchLength 4 -min 20).
Reads were subsampled using SeqKit's sample v2.5.1 to acheieve equal read depth.
Reads were aligned to the the appropriate reference genome using STAR v2.7.10a for humans and Hisat2 v2.2.1 for the livestock.
Alignment files were converted to tag directories using HOMER2 batchMakeTagDirectory.pl.
BedGraph files were generated from the tag directories using makeUCSCfile.
Peaks representing Transcription Start Regions (TSRs) for csRNA-seq and expressed small RNAs for sRNA-seq, were defined using HOMER’s findcsRNATSR.pl and findPeaks, respectively. A minimum read count of 10 per 10 million was required for regions to be considered.
Assembly: hg38, GCF_002263795.2, modified_GCA_018282365.1
Supplementary files format and content: BedGraph files (.bedGraph.gz) containing the locations that denote TSS positions.
Supplementary files format and content: Text files (.tss.txt) containing tab-delimited data on identified transcription start site clusters (TSRs).
Supplementary files format and content: Modified gtf file that underwent an ID update to achieve consistency with the fasta genome.
 
Submission date May 20, 2024
Last update date Jun 26, 2024
Contact name Sascha Duttke
Organization name Washington State University
Department School of Molecular Biosciences
Lab Dr. Duttke Lab
Street address BLS 147, 100 Dairy Road
City Pullman
State/province Washington
ZIP/Postal code 99164
Country USA
 
Platform ID GPL24676
Series (1)
GSE267848 Efficient small DNA fragment sequencing and miRNA, small RNA or csRNA-seq libraries using AVITI
Relations
BioSample SAMN41455496
SRA SRX24605984

Supplementary file Size Download File type/resource
GSM8279792_Homo_sapiens-A375_Illumina_sRNA-r2.bedGraph.gz 1.4 Mb (ftp)(http) BEDGRAPH
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Raw data are available in SRA

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