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| Status |
Public on May 25, 2020 |
| Title |
NXF8_nuc |
| Sample type |
SRA |
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| Source name |
CytoLib, siNXF1 nuclear fraction, rep4
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| Organism |
Homo sapiens |
| Characteristics |
tissue: MCF-7 cell line
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| Growth protocol |
MCF7 cells (ATCC) were grown in DMEM (Gibco) containing 10% fetal bovine serum and Penicillin-Streptomycin mixture (1%) at 37°C in a humidified incubator with 5% CO2. For export factor knockdown, cells were transfected with 10nM siRNA pool targeting indicated genes or with a control pool (Dharmacon) using Lipofectamin 3000 reagent (L3000001, Thermo Fisher). For double knockdowns, 5nM of each pool were used for a total mix of 10nM. Cells were collected for cytosolic and nuclear fractionation 72h post-transfection for all export factor depletion assays, except for siNXF1 and siALY+UAP56, where we collected the cells 48h post-transfection.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were washed in cold PBS and detached from plates by 10mM EDTA, a fraction was transferred to a new tube and RNA was extracted with TRIREAGENT (MRC) to obtain WCE. Remaining cells were washed in cold PBS, resuspended in 180µl RLN buffer (50mM Tris•Cl pH8, 140mM NaCl, 1.5mM MgCl2, 10mM EDTA, 1mM DTT, 0.5% NP-40, 10U/ml RNAse inhibitor), and incubated on ice for 5 min. The extract was centrifuged for 5min at 300g in a cold centrifuge, the supernatant was transferred to a new tube and centrifuged again for 1min at 500g in a cold centrifuge. The supernatant (cytoplasmic fraction) was transferred to a new tube and RNA was extracted using TRIREAGENT. The nuclear pellet was washed once in 180µl RLN buffer, resuspended in 1ml of buffer S1 (250mM Sucrose, 10mM MgCl2, 10U/ml RNAse inhibitor), layered over 3ml of buffer S3 (880mM Sucrose, 0.5mM MgCl2, 10U/ml RNAse inhibitor), and centrifuged for 10min at 2800g in a cold centrifuge. The supernatant was removed and RNA was extracted from the nuclear pellet using TRIREAGENT.
One microgram of RNA was used for cDNA production using the qScript Flex cDNA synthesis kit (95049, Quanta) and a gene specific primer containing part of the Illumina RD2 region. The entire cDNA reaction was diluted into 100 μl second strand reaction with a primer containing a unique molecular identifier (UMI) and part of the Illumina RD1 region. The second strand reaction was carried for a single cycle using Phusion Hot Start Flex DNA Polymerase (NEB, M0535), purified using AMpure beads at a 1.2:1 beads:sample ratio and eluted in 20μl ddH2O. 20µl of the second strand reaction was used for amplification with barcoded primers, and the amplified libraries were purified by two-sided AMpure purification; First with a 0.5:1 beads:sample ratio followed by a 0.7:1 ratio.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Library strategy: MPRA
Quantification using RSEM
Alignment to the hg19 human genome using STAR
Preparation of bigWig files using wigToBigWIg
Genome_build: hg19
Supplementary_files_format_and_content: RSEM quantification files and bigWig read coverage, summary files for half-lives estimated for SLAM-seq and UMI count for MPRA data
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| Submission date |
Oct 21, 2019 |
| Last update date |
May 26, 2020 |
| Contact name |
Igor Ulitsky |
| E-mail(s) |
igor.ulitsky@weizmann.ac.il
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| Organization name |
Weizmann Institute of Science
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| Street address |
Hertzl St.
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| City |
Rehovot |
| ZIP/Postal code |
76100 |
| Country |
Israel |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE139151 |
Gene architecture and sequence composition underpin selective dependency of long RNAs on components of the nuclear export pathway |
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| Relations |
| BioSample |
SAMN13067682 |
| SRA |
SRX7027818 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4132178_NXF8_nuc.combined.txt.gz |
5.2 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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