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Sample GSM6241104 Query DataSets for GSM6241104
Status Public on May 09, 2023
Title ZFRlong/ILF2-80 nM
Sample type SRA
 
Source name Protein-bound RNA
Organism synthetic construct
Characteristics sample type: synthetic sequence library
treatment: ZFRlong/ILF2-80 nM
molecule subtype: Protein-bound RNA
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.
 
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina HiSeq 3000
 
Description ZFR_long_ILF2_RBNS.csv
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.
 
Submission date Jun 14, 2022
Last update date May 09, 2023
Contact name J. Robert Hogg
E-mail(s) j.hogg@nih.gov
Organization name National Heart, Lung, and Blood Institute
Department Biochemistry and Biophysics Center
Street address 50 South Drive, Room 2341
City Bethesda
State/province MD
ZIP/Postal code 20892
Country USA
 
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.
Relations
BioSample SAMN29024450
SRA SRX15696425

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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