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Status |
Public on Jan 17, 2024 |
Title |
FLASH HSPA8 rep1 |
Sample type |
SRA |
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Source name |
HEK293 T-Rex
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Organism |
Homo sapiens |
Characteristics |
cell line: HEK293 Flp-In T-Rex cells
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Treatment protocol |
Expression of 3xFLAG-HBH tagged proteins was induced with 1 μg/ml tetracycline (Sigma-Aldrich, T7660)
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Growth protocol |
HEK293 Flp-In T-Rex cells were cultured in DMEM high glucose medium supplemented with 10% FBS (Sigma-Aldrich F0804), 2mM L-glutamine (Sigma-Aldrich, G7513) and 1% penicillin-streptomycin (Sigma-Aldrich, P4333). Cells were kept at 37 °C in 5% CO2 incubator. All cell lines were routinely checked for mycoplasma contamination by PCR.
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Extracted molecule |
total RNA |
Extraction protocol |
FLASH-seq protocol was applied as described in Aktaş et al., 2017. Briefly, cells were crosslinked with 0.2mJ/cm2 UV-C irradiation, lysed with 1xNLB and homogenized by waterbath-sonication. Target protein of interest were affinity purified in tandem with nickel-charged paramagnetic beads and streptavidin-coupled paramagnetic beads against the tagged protein of interest. After a brief RNAseI-digestion, RNA 3'-ends were healed with T4 PNK. Custom-made, barcoded adapters were ligated using T4 RNA Ligase 1, for 1 hr at 25˚C. Custom FLASH adapters contained two barcodes and random nucleotides adjacent to the 3'-adapters according to the pattern NNBBNTTTTTTNN (N: random tag nucleotide, T: tag nucleotide, B: RY-space tag nucleotide). Random tags were used to merge PCR-duplicates, regular tags were used to specify the pulldown condition, and semi-random RY-space tags were used to distinguish the biological replicates (RR: replicate A, YY: replicate B, R: purine, Y: pyrimidine). Excess adapters were washed away and RNA was isolated with Proteinase K treatment and column purification. Isolated RNA was reverse-transcribed and RNaseH-treated. cDNA was column-purified and circularized with CircLigase for 16hrs. Circularized cDNA was directly PCR amplified, quantified with Qubit / Bioanalyzer and sequenced on Illumina NextSeq 500 in paired-end mode. Libraries were prepared according to NEBNext Ultra II DNA Library preparation kit (NEB#E7645S)
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Library strategy |
RIP-Seq |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
Illumina HiSeq 3000 |
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Data processing |
Demultiplexing (flexbar, version 3.3) and UMI trimming and header labelling (UMITools, Version 0.5.1) Adapter trimming, quality control and demultiplexing barcodes (only R2) (trim_galore (Version 0.4.4) --stringency 3 -q 30 --paired --retain_unpaired -r1 15 -r2 15 --fastqc --three_prime_clip_R2 13 --length 14 --clip_R1 6 ) Paired-end reads were merged using bbmerge from bbmap (Version 37.54) Genome alignment, allowing up to 1000 mapping locations (STAR (Version 2.6.0b) --outFilterMultimapNmax 150 --outFilterScoreMinOverLread 0 --outFilterMatchNminOverLread 0 --outFilterMatchNmin 0 --alignEndsType EndToEnd --alignIntronMax 100000) Duplicated reads into individual crosslinking events with UMITools (Version 0.5.1) Peak calling (yodel.py (1.0) --mph 5) and alternative contigs removal (GNU Awk 4.0.2) Bigwigs were generated using bamCoverage from deeptools2 Assembly: GRCH38 Supplementary files format and content: BED File (yodel output) and bigwig (normalisation: None)
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Submission date |
Apr 23, 2023 |
Last update date |
Jan 17, 2024 |
Contact name |
Andrés Manuel Herrero-Ruiz |
E-mail(s) |
ah2192@cam.ac.uk
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Organization name |
MRC-University of Cambridge
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Department |
MRC Toxicology Unit
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Lab |
Ritwick Sawarkar's Laboratory
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Street address |
Gleeson Building, Tennis Court Road
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City |
Cambridge |
ZIP/Postal code |
CB2 1QR |
Country |
United Kingdom |
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Platform ID |
GPL21290 |
Series (2) |
GSE191242 |
HSP70 binds RNA and regulates transcription by RNA Polymerase III [FLASH-seq] |
GSE191245 |
HSP70 binds to specific non-coding RNA and regulates human RNA polymerase III |
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Relations |
BioSample |
SAMN34338399 |
SRA |
SRX20078055 |
Supplementary file |
Size |
Download |
File type/resource |
GSM7221175_FLASH_HSPA8_rep1.bw |
2.0 Mb |
(ftp)(http) |
BW |
GSM7221175_FLASH_HSPA8_rep1_peaks.bed.gz |
62.2 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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