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| Status |
Public on Feb 19, 2021 |
| Title |
RNA-Seq (+)Harringtonine bio_rep1 tech_rep1 |
| Sample type |
SRA |
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| Source name |
HEK293 (+) Harringtonine
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| Organism |
Homo sapiens |
| Characteristics |
cell line background: HEK293
treatment: Harringtonine (2 ng/mL), one hour
cellular fraction: Whole cell
biological replicate: 1
technical replicate: 1
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| Treatment protocol |
Cells were subjected (+) or not (–) to a one hour treatment with harringtonine (2 ng/mL) prior to harvesting.
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| Growth protocol |
HEK293 cells were grown at 37C in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/ streptomycin.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated in TRIzol using the Direct-zol RNA Kit (Zymo Research, R2062).
Deep sequencing libraries were prepared from three biological replicates per condition using the KAPA RNA HyperPrep Kit with RiboErase (HMR) (Roche, 08098131702) with a modified protocol as follows. Isolated RNA (7 ug; Quantified using Qubit RNA Broad Range Assay Kit (Thermo Fischer Scientific, Q10210) was first treated with Turbo DNase using the standard protocol (Thermo Fischer Scientific, AM2239). Treated RNA (1 ug) was then used as input at the rRNA depletion step in the KAPA kit protocol. To generate 200-550 nt fragments, fragmentation was carried out for 5 minutes at 94°C and samples immediately chilled on ice. Following seven amplification cycles using the dual index adapters (5 uL of a 1.5 uM per uL dilution; final concentration of 10 nM) from the KAPA Dual-Indexed Adapter Kit (KK8722) (Roche, 08278555702), PCR products containing 200 to 550 nt inserts were size selected using SPRIselect beads (Beckman Coulter Life Sciences, B23318) and quantified using the Qubit dsDNA Broad Range Assay Kit (Thermo Fischer Scientific, Q32850). As each library contained a unique barcode, all libraries (3 biological replicates + or - harringtonine treatment) were mixed and sequenced on an Illumina NextSeq 550 instrument using the High Output Kit v2.5 (Illumina, Inc., 20024908). All libraries were loaded at 1.8 pm with 5% PhiX and all data was written to BaseSpace (Illumina, Inc.).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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| Description |
RS_PlusHarr_Brep1_Trep1
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| Data processing |
Prior to alignment, adaptor sequences and long stretches (≥ 20 nt) of adenosines were trimmed from read 3′ ends.
All libraries were filtered for reads aligning to repeat regions, as defined by RepeatMasker, using STAR v2.5.3a.
Remaining reads were aligned with STAR on two-pass mode allowing a maximum of 3 mismatches per pair and highly penalized deletions and insertions.
Mapped reads were then filtered for low mapping quality (MAPQ < 5) and/or duplicated reads, identified with the MarkDuplicates tool (Picard v2.17.8).
Genome_build: hg38
Supplementary_files_format_and_content: bigWig files containing mapped and filtered reads
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| Submission date |
May 10, 2020 |
| Last update date |
Feb 19, 2021 |
| Contact name |
Carrie Kovalak |
| E-mail(s) |
[email protected]
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| Organization name |
University of Massachusetts Medical School
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| Department |
RNA Therapeutics Institute
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| Lab |
Melissa Moore
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| Street address |
365 Plantation Street
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| City |
Worcester |
| State/province |
MA |
| ZIP/Postal code |
01605 |
| Country |
USA |
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| Platform ID |
GPL21697 |
| Series (1) |
| GSE150228 |
Deep sequencing of pre-translational mRNPs reveals hidden flux through evolutionarily conserved AS-NMD pathways |
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| Relations |
| BioSample |
SAMN14883613 |
| SRA |
SRX8300325 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4543722_RS_PlusHarr_Brep1_Trep1.bw |
233.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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