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Status |
Public on May 09, 2023 |
Title |
ZFRlong/ILF2-80 nM |
Sample type |
SRA |
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Source name |
Protein-bound RNA
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Organism |
synthetic construct |
Characteristics |
sample type: synthetic sequence library treatment: ZFRlong/ILF2-80 nM molecule subtype: Protein-bound RNA
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Extracted molecule |
total RNA |
Extraction protocol |
The DNA template for T7 transcription of the RBNS library containing 40 random nucleotides was synthesized by Integrated DNA Technologies and transcribed with the MEGAshortscript T7 transcription kit (ThermoFisher Scientific). Four concentrations of each ZFRDZF/ILF2, ZFRlong/ILF2 (5, 20, 80, and 320 nM) or two concentrations of control protein PTBP1 (20 and 80 nM) were equilibrated in 45 µl binding buffer (20 mM HEPES pH 7.5, 150 mM potassium acetate, 3 mM magnesium acetate, 0.01% Tween-20, 1 mM DTT, 1 mM zinc sulfate, 0.5 mg/ml BSA) for 30 minutes at RT, and 90 µl 1 µM RBNS RNA library supplemented with 20 units of SUPERaseIn (ThermoFisher Scientific) was added and rotated for 1 hr at RT. Pre-washed 25 µl His-Tag Dynabeads (ThermoFisher Scientific) were added, rotated for 1 hour at RT, and washed three times with wash buffer (20 mM HEPES pH 7.5, 150 mM potassium acetate, 0.01% Tween, 0.5 mM EDTA, 0.5 mg/ml BSA). Bound RNA was eluted with 100 µl elution buffer (10 mM Tris, pH 7.0, 1 mM EDTA, 1% SDS) by heating at 70°C for 10 min. Eluted RNA was purified with Zymo RNA Clean & Concentrator-5 columns (Zymo Research), reverse-transcribed with SuperScript IV (ThermoFisher Scientific), PCR amplified with AccuPrime PFX SuperMix (ThermoFisher Scientific), and sequenced on the Illumina HiSeq 3000 platform.
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Library strategy |
OTHER |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
Illumina HiSeq 3000 |
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Description |
ZFR_long_ILF2_RBNS.csv
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Data processing |
library strategy: RNA Bind-n-Seq RBNS data were analyzed using rbns_pipeline software (https://github.com/alexrson/rbns_pipeline). Supplementary files format and content: Comma-delimited files contain B values generated by the RBNS analysis pipeline for the indicated 6-mer sequences.
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Submission date |
Jun 14, 2022 |
Last update date |
May 09, 2023 |
Contact name |
J. Robert Hogg |
E-mail(s) |
j.hogg@nih.gov
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Organization name |
National Heart, Lung, and Blood Institute
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Department |
Biochemistry and Biophysics Center
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Street address |
50 South Drive, Room 2341
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City |
Bethesda |
State/province |
MD |
ZIP/Postal code |
20892 |
Country |
USA |
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Platform ID |
GPL27484 |
Series (2) |
GSE206060 |
A network of DZF proteins controls alternative splicing regulation and fidelity [RNA Bind-n-Seq] |
GSE206061 |
A network of DZF proteins controls alternative splicing regulation and fidelity. |
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Relations |
BioSample |
SAMN29024450 |
SRA |
SRX15696425 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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