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| Status |
Public on Jan 17, 2024 |
| Title |
FLASH HSPA1A rep2 |
| 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 |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
The second replicate of FLASH-seq experiment was only used to identify common peaks. Both BED files are uploaded. However, only replicate 1 signal (bigwig) is shown along the paper.
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| Data processing |
Library strategy: FLASH-seq
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
Genome_build: GRCH38
Supplementary_files_format_and_content: BED File (yodel output) and bigwig (normalisation: None)
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| Submission date |
Dec 20, 2021 |
| 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 |
GPL18573 |
| 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 |
SAMN24224827 |
| SRA |
SRX13453797 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5742142_FLASH_HSPA1A_rep2_peaks.bed.gz |
40.1 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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