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Sample GSM5452294 Query DataSets for GSM5452294
Status Public on Apr 01, 2022
Title PL_SNU_WT
Sample type SRA
 
Source name Human Embryonic Kidney
Organism Homo sapiens
Characteristics cell line: HEK293
treatment: Untreated
protocol: SNU-Seq
fraction: Nascent RNA
Treatment protocol For SNU-Seq labelling, 60 million cells per sample were incubated with 500 µM 4sU for 10 mins
Growth protocol HeLa and HEK293 cells were cultured in DMEM supplemented with 10 % (v/v) FBS and 1 % (v/v) Penicillin-Streptomycin. The incubator was set at 37 ◦C at 5 % CO2. Cells were passaged 24 hours before harvesting for next-generation sequencing ex- periments at which point they were grown to 80 % confluency. Cells were counted on a Nexcelom Biosciences Auto 2000 Cell Counter
Extracted molecule total RNA
Extraction protocol Cells were washed rapidly in PBS before adding 0.7 mL QIAZOL per 10 million cells. The cells were scraped off into 2mL Eppendorffs, vortexed for 10 s and placed on ice for 10 minutes. If required for comparative calibration, 2.4 ng of thio-labelled RNA spike-ins per 1 million cells were added at this stage. Total RNA was purified by adding 320 µL chloroform, vortexing for 10 s, incubating at room temperature for 3 minutes, and centrifugation at 4◦C for 15 minutes at 12000 rpm. The upper phase was transferred to a new Eppendorf with 800 uL isopropanol. The sample was left at room temperature for 5 minutes before centrifugation at 4 ◦C for 15 minutes at 15000 rpm. After removing the supernatant, the pellet was washed 3 times in 320 uL 75% ethanol (prepared freshly), with centrifugation at 4 ◦C for 15 minutes at 15000 rpm. The pellet was then air-dried for 10 minutes before dissolving in 160 µL RNAse-free water. At this stage samples were frozen at -80 ◦C. RNA amounts were checked with Nanodrop. 300 µg of total RNA in 400 µL water were chilled on ice for 10 minutes. Samples were split equally into two Eppendorfs, incubated at 65 ◦C for 10 minutes, and then on ice for 5 minutes. After quick-spinning, 300 µL RNase-free water, 100 µL 10x Biotinylation Buffer (100 mM Tris pH 7.5, 1m mM EDTA pH 8.0), 200 µL DMF, and 200 mL EZ-link HPDP Biotin in DMF (1mg / mL, stored in the dark at 4 ◦C). Incubate in the dark at room temperature for 2 hours with shaking at 800 rpm. RNA was purified using Phase-Lock tubes and resuspended in 100 µL RNase-free water. Samples were pooled back together. Following purification, the RNA was not sonicated but carried through to biotinylation and streptavidin purification.For thio-labelled RNA separation, a wash buffer (100 mM Tris pH 7.5, 10 mM EDTA pH 8.0, 1 M NaCl, 0.1 % Tween-20) was prepared freshly – one kept at room temperature (RT-WB), and one at 65 ◦C (65-WB). 100 µL uMACS streptavidin beads were added to 200 µL RNA-Biotin and incubated in a thermomixer at 4 ◦C for 15 minutes at 800 rpm. During this time, the uMACS magnetic stand and rack were prepared alongside a drainer. uMACS columns were placed into the magnetic rack and equilibrated with 900 µL RT-WB for 10 minutes.The biotin-RNA-bead mix was added to the uMACS column. The flow-through was collected and re-loaded to the columns. The second flow-through was discarded. Columns were washed 3 times with 900 µL of 65-WB and then 3 times with 900 µL RT- WB. Elution of labelled RNA was carried out with 100 µL 100 mM DTT. After 5 minutes, elution was performed again with an additional 100 uL 100 mM DTT. Samples were then immediately carried through for RNA purification using the miRNeasy kit. RNA was eluted with 15 µL RNase-free water. Quality and quantity of thiolabelled RNA was checked using the Agilent Bioanalyzer with an RNA pico chip. RNAs were polyadenylated using the NEB Poly A Polymerase kit following the kit instructions. The reaction with 150 ng thiolabeled RNA was left for 45 minutes at 37 ◦C before isopropanol precipitation. The pellet was resuspended in 11 µL RNase-free water. Qualities and amounts were checked on the Qbit fluoremeter and Agilent bioanalyzer.
Libraries were prepared following the Quant-Seq Lexogen 3’ mRNA kit instructions with 13 PCR cycles. Following the manufacturer’s instructions, the chips loaded by the Ion Chef were then sequenced on an Ion Proton Sequencing platform
Thiolabelled spike-ins were generated by amplifying 3 1000-bp long DNA frag- ments from genomic yeast DNA with a 42 % GC content, covering the genes bat2, hxt1, and gal1 using forward primers containing the T7 promoter sequence. The PCR product was purified and then transcribed in vitro following the MEGAscript T7 tran- scription kit instructions, using a thio-UTP to UTP ratio of 1:5.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Ion Torrent Proton
 
Description Sample 1 (PL_SNU_WT repeat 1), aka 17
Sample 2 (PL_SNU_WT repeat2), aka 18
Data processing After quality control with fastqc, single-end fastq files were trimmed with Trimmomatic (Bolger et al., 2014) to remove reads with a quality score below 20 in a sliding window of 5 bp. The poly(A) signals were removed with the clip option. Sequences were then aligned with hisat2 with the same settings as for TT-Seq. Sorted bam files were generated using samtools. Calibration of samples was achieved by calculating scaling factors of spike-in counts between samples based on counts tables generated by the bioconductor package featureCounts. Bedgraph and bigwig files were generated from bam files using bedtools, and wigTobigWig (UCSC Genome Browser), respectively. 3’end single basepair resolution was achieved by using the bedtools -bg -3 option. In order to remove potential signals arising from internal priming due to intragenic poly-A stretches, peak-calling on the SNU-Seq bedgraph files (separately for each strand) was performed using macs2 with the narrowpeaks and no-model options. DNA sequences of these peaks were retrieved using the bedtools getfasta command before generating a bed file containing all peak coordinates with sequences containing at least 8 consecutive As (or Ts on the reverse strand). Finally, the ’bedtools intersect -wa’ option was used to filter out any SNU-Seq signals that lie within these peaks.
genome build: hg38
Supplementary_files_format_and_content: The six processed files report (i) the complete SNU-seq signal generated by sequencing from the artificial polyA tract on both the forward and reverse strand, (ii) the 3' end reads only to yield single nucleotide resolution data on both the forward and reverse strand (the standard SNU-seq readout) and (iii) the same 3' end single nucleotide resolution data but lacking any signal from internal priming at polyA tracts 8nt or longer which was removed bioinformatically on both the forward and reverse strands.
 
Submission date Jul 14, 2021
Last update date Apr 01, 2022
Contact name Jane Mellor
E-mail(s) Jane.mellor@bioch.ox.ac.uk
Phone 07810544459
Organization name University of Oxford
Department Biochemistry
Lab Mellor
Street address South Parks Road
City Oxford
ZIP/Postal code OX1 3QU
Country United Kingdom
 
Platform ID GPL17303
Series (1)
GSE179306 Mapping Human Transient Transcriptomes Using Single Nucleotide Resolution 4sU Sequencing (SNU-Seq)
Relations
BioSample SAMN20208730
SRA SRX11439176

Supplementary file Size Download File type/resource
GSM5452294_PL_SNU_WT_merged_3pr_F.bedgraph.gz 27.8 Mb (ftp)(http) BEDGRAPH
GSM5452294_PL_SNU_WT_merged_3pr_R.bedgraph.gz 21.9 Mb (ftp)(http) BEDGRAPH
GSM5452294_PL_SNU_WT_merged_F.bedgraph.gz 49.0 Mb (ftp)(http) BEDGRAPH
GSM5452294_PL_SNU_WT_merged_R.bedgraph.gz 39.3 Mb (ftp)(http) BEDGRAPH
GSM5452294_SNU_WT_polyA_cleanup_sorted_fwd.bedgraph.gz 13.1 Mb (ftp)(http) BEDGRAPH
GSM5452294_SNU_WT_polyA_cleanup_sorted_rev.bedgraph.gz 11.5 Mb (ftp)(http) BEDGRAPH
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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