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Sample GSM5452295 Query DataSets for GSM5452295
Status Public on Apr 01, 2022
Title PL_size_selected_SNU_WT
Sample type SRA
 
Source name Human Embryonic Kidney
Organism Homo sapiens
Characteristics cell line: HEK293
treatment: Untreated
protocol: size-selected SNU-Seq
fraction: Nascent RNA
Treatment protocol For ssSNU-Seq, sufficient cells were incubated with 500 µM 4sU for 10 mins to enable purification of 250ng of nascent thiolabelled RNA.
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 Nascent, thio-labelled RNA was generated as described for SNU-Seq without size selection. The nascent RNA was then run on a 3.5 % TBE-Urea gel (8M urea). For this, 250 ng of nascent RNA was mixed with 2x loading dye and boiled for 2 minutes at 80 ◦C. The gel was kept on ice and pre-run for 10 minutes at 80 V, using ice-cold ultra-pure TBE (1x) as the running buffer. Wells of the gel were resuspended with TBE before loading samples. Samples were then run at 80 V for 90 minutes. The gel was transferred onto a square petri dish and incubated in 1x ultra-pure TBE with SYBR Gold (1/10,000 v/v) for 5 minutes. The gel was rinsed twice in 1x ultra-pure TBE before imaging. For purification of the small nascent RNA fraction, the gel was incubated at -80◦C for 10 minutes. The small band of 70-100 bp size was cut using a razor blade. RNA was extracted from the gel slice using dialysis: The gel slice, along with 0.8 mL 10 mM Tris-HCl (pH 7), was placed into SnakeSkin dialysis membrane sealed by two Eppendorf caps. The dialysis was run for 35 minutes at 80 V. The RNA was then purified with isopropanol precipitation and eluted in 11 µL RNase-free water. Quality and size distribution of the size-selected RNA was checked on the Agilent Bioanalyzer using an RNA pico chip. The size selected nascent RNA was treated with 5’ Pyrophosphohydrolase (NEB) in ThermoPolQR reaction buffer following the manufacturer’s instructions. The reaction was stopped with 1 µL 500 mM EDTA and heat-inactivated by incubation at 65 ◦C for 5 minutes. RNA was again purified with isopropanol precipitation and resuspended in 6 µL RNase-free water.
The NEBNext Small RNA Library kit for Illumina following the kit instructions. Library quality was assessed with the Agilent Bioanalyzer using a DNA High Sensitivity chip. Sequencing was performed on an Illumina NextSeq 500 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 Illumina NextSeq 500
 
Description Sample 3
Data processing Fastq files were quality-trimmed with Trim-Galore and aligned with bowtie2. Sorted bam files were generated from aligned sam files using samtools with a filtering step added to only retain reads that have a mapping quality score above 30 (-q 30). As above, 3’ end single basepair resolution was achieved by using the bedtools -bg -3 option when generating bedgraph files.
To generate TSS annotations from the size-selected SNU-Seq samples, 5’ end coverage was generated using the -bg -5 option (instead of -3) when generating bedgraphs from bam files in bedtools. To generate 5’end reads that only occur in nucleosome-depleted regions, ATAC-Seq data in HEK293 cells E-MTAB-6195) were used. The ATAC-Seq fastq files were trimmed with Trim-Galore, aligned with bowtie2, and converted to bam and bed files using samtools and bedtools, respectively.Subsequently, macs2 was used to call peaks (no model assumed). The intersect option in bedtools was then used to retain only those 5’end SNU-Seq (size-selected) signals that are located within ATAC-Seq peaks. The resulting bedgraph files were then used as an input to identify TSS cluster centres using Paraclu which identifies clusters in a sliding window, and a cluster value threshold of 30 was applied. Cluster-centres were calculated using the mean position of the cluster, and TSS candidates were verified in Matlab as follows: Only those TSS candidates were assigned as true TSSs where the summed TT-seq signal (using HEK WT 10 minute- labelled TT-Seq) in the 100 bp downstream of the candidate TSS was at least 5 times greater than in the 100 bp upstream of the candidate TSS. TSScall, a python code, was then used to identify annotated and un-annotated (novel) TSSs using the comprehensive Gencode (v29) annotation file which yielded 2955 previously annotated TSSs and 1428 novel transcription start sites. The read threshold for this was set to 5.
genome build: hg38
Supplementary_files_format_and_content: The four processed files report 5' end reads on the forward and reverse strand and 3' end reads on the forward and reverse strands from the short size selected thiolabelled nascent RNA.
 
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 GPL18573
Series (1)
GSE179306 Mapping Human Transient Transcriptomes Using Single Nucleotide Resolution 4sU Sequencing (SNU-Seq)
Relations
BioSample SAMN20208729
SRA SRX11439177

Supplementary file Size Download File type/resource
GSM5452295_HEK_FII_3_F.bedgraph.gz 3.6 Mb (ftp)(http) BEDGRAPH
GSM5452295_HEK_FII_3_R.bedgraph.gz 3.3 Mb (ftp)(http) BEDGRAPH
GSM5452295_HEK_FII_5_F.bedgraph.gz 3.3 Mb (ftp)(http) BEDGRAPH
GSM5452295_HEK_FII_5_R.bedgraph.gz 3.0 Mb (ftp)(http) BEDGRAPH
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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