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Sample GSM4559593 Query DataSets for GSM4559593
Status Public on Oct 06, 2020
Title 293T Control replicate 3
Sample type SRA
 
Source name embryonic kidney
Organism Homo sapiens
Characteristics cell type: embryonic kidney
cell line: 293T
rna fraction: total RNA
genotype/variation: Nontargeting siRNA
spike in: 1 uL of a 1:10 dilution of ERCC spike RNAs (Invitrogen) was added to 1 million 293T cells resuspended in Trizol
Treatment protocol Nontargeting siRNA treatment (48 hr)
Growth protocol 293T cells were grown at 37°C with 5% CO2 in DMEM, supplemented with 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin.
Extracted molecule total RNA
Extraction protocol 293T cells were harvested and washed with PBS. Cells were counted, and 1 x 106 cells were resuspended in 1mL of Trizol and spiked with 1 µL of 1:10 diluted ERCC Spike-in Mix (Invitrogen) and RNA was purified.
1 µg of RNA from each sample was diluted in 10uL of water. The input RNA was subjected to RNA-seq library generation using the TruSeq Stranded Total RNA sequencing kit with RiboZero rRNA depletion (Illumina). The final libraries were amplified to 10 cycles and purified using AMPure XP beads (Beckman Coulter).
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 4000
 
Description ribo-depleted Total RNA isolated from 293T cells treated with control siiRNA
Adelman_hg38_293T_Control_RNAseq_N3_plus.bw
Adelman_hg38_293T_Control_RNAseq_N3_minus.bw
Data processing PRO-seq: Paired-end reads were trimmed to 40nt, for adaptor sequence and low quality 3' ends using cutadapt 1.14, discarding those containing reads shorter than 20nt (-m 20 -q 10); RNA-seq: Reads were filtered to require a mean quality score ≥ 20, trimmed to 100 nt, and mapped to the hg38 human genome assembly using STAR.
PRO-seq: Remaining pairs were paired-end aligned (using Bowtie 1.2.2) to the hg38 spike genome index to determine spike-normalization ratios based on uniquely mapped reads. Mappable pairs were omitted from subsequent analysis; RNA-seq: Default parameters were used, except that multimappers were randomly assigned (outMultimapperOrder Random), spurious junctions were filtered (outFilterType BySJout), minimum overhang for non-annotated junctions was set to 8 nt (alignSJoverhangMin 8), and non-canonical alignments were removed (outFilterIntronMotifs RemoveNoncanonicalUnannotated).
PRO-seq: Remaining unmapped pairs were aligned to the dm3 genome assembly. Identical parameters were used in each alignment described: up to 2 mismatches, and uniquely mappable and unmappable pairs routed to separate output files (-v2, -un, --best).
PRO-seq: Pairs mapping to dm3, representing biotin-labeled RNA 3' ends, were separated, and strand-specific counts of the 3' mapping positions determined at single nucleotide resolution, genome-wide, and expressed in bedGraph format with "plus" and "minus" strand labels swapped for each 3' bedGraph, to correct for the "forward/reverse" nature of Illumina paired-end sequencing.
PRO-seq: Based on similar recovery of spike-in reads, depth normalization was performed and bedGraphs from replicates of each condition were combined by summing counts per nucleotide.
Genome_build: dm3 (PRO-seq, RNA-seq); hg38 (RNA-seq)
Supplementary_files_format_and_content: PRO-seq: bedGraph containing combined count of end 1 3' mapping locations for all replicates for a given treatment; RNA-seq: bigWig containing coverage of all replicates for a given treatment
 
Submission date May 19, 2020
Last update date Oct 06, 2020
Contact name Karen Adelman
E-mail(s) karen_adelman@hms.harvard.edu
Organization name Harvard Medical School
Department Biological Chemistry and Molecular Pharmacology
Street address 45 Shattuck St. LHRRB-201a
City Boston
State/province MA
ZIP/Postal code 02115
Country USA
 
Platform ID GPL20301
Series (1)
GSE150844 Integrator Recruits Protein Phosphatase 2A to Prevent Pause Release and Enable Transcription Termination
Relations
BioSample SAMN14971917
SRA SRX8363232

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

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