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| Status |
Public on Nov 07, 2023 |
| Title |
HEK293,H1.2KO |
| Sample type |
SRA |
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| Source name |
HEK293
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293
cell type: Human embryonic kidney
genotype: H1.2 knockout
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| Extracted molecule |
total RNA |
| Extraction protocol |
For library generation, 1 x 106 cells were spiked with 5% permeabilizedDrosophilaS2 cells for data normalization and used as input for PRO-seq. Nascent RNA was labeled through a biotin-NTP run-on as follows: Permeabilized cells were added to an equal volume of a 2X run-on reaction mix. and incubated at 30 °C for 5 min. RNA was isolated using TRIzol Reagent. Isolated RNA was fragmented by base hydrolysis with 0.25 N NaOH for 9 min on ice, followed by neutralization with one volume of 1 M Tris-HCl, pH 6.8. To select for nascent RNA, 48 μL of washed Streptavidin M-280 magnetic beads in binding buffer were added to the fragmented RNA, and samples were rotated at room temperature for 20 min. The Streptavidin M-280 magnetic beads were washed twice in each of the following three buffers: high salt buffer , binding buffer, and low salt buffer. Beads were resuspended in TRIzol LS Reagent and heated at 65 °C for 5 min, cooled to room temperature, and heated at 65 °C for 5 min again to elute the RNA from the beads.
Nascent RNA was resuspended in 10 µM of the VRA3 3′ end adapter in T4 Ligase buffer and was 3′ end ligated with T4 RNA ligase I for 2 h at room temperature. The sample was enriched for ligated nascent RNAs by binding to Streptavidin M-280 magnetic beads as described above. The beads were washed twice in high, binding, and low salt buffers, then once in 1X ThermoPol Buffer. To prepare the nascent RNA for 5′ end adapter ligation, the 5′ ends of the RNA were decapped using RppH suspended in RppH buffer, then 5′ end was recapped with RNA 5′ pyrophosphohydrolase at 37 °C for 1 h in T4 ligase buffer. Beads were then washed in high salt buffer, low salt buffer, and 1X T4 PNK Reaction Buffer. Samples were treated with T4Poly-nucleotideKinase for 1 h at 37 °C, and the reverse 5′ RNA adapter VRA5 was ligated to the RNA by incubation with T4 RNA ligase I for 2 h at room temperature. Following the 5′ end ligation, beads were washed twice in high, binding, and low salt buffers, then once in 0.25X FSS Buffer . Reverse transcription was performed using Superscript IV Reverse Transcriptase with 25 pmol of the Illumina TRU-seqRP1Primer. The RT product was eluted from the beads by heating the samples twice at 95°C for 30 s. All libraries were amplified by 12 cycles of PCR with 12.5 pmol of Illumina TRU-seq RPI-index primers, excess RP1 primer, and Q5 DNA Polymerase. The samples were sequenced using a HiSeq instrument with a mid-output 150bp cycle run.
Six nucleotide Barcode specific primers were used in order to isolate the results from each of the specific samples (WT, 1-KO, 2-KO, 4-KO and 5-KO).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Cutadapt (v3.4, https://doi.org/10.14806/ej.17.1.200) was used to trim adapter sequences from paired-end reads and to remove low-quality bases and reads shorter than 20 nt with -m20 -q 30,30.
Processed reads were first aligned to the Drosophila genome assembly dm3 using Bowtie2 v1.2.3 with --trim5 6 -p 20 --score-min C,-13,0.
To perform spike-in normalization in all samples, we counted the primary aligned mapped reads to the dm3 genome assembly using samtools view (v1.12) with the -c and -F260 options.
Paired reads that did not align to dm3, were aligned to the hg19 genome assembly using STAR v2.7.10b with the following parameters: --outMultimapperOrder Random --outFilterIntronMotifs RemoveNoncanonicalUnannotated --outFilterType BySJout --outSAMtype BAM SortedByCoordinate --alignSJoverhangMin 8 –outFileNamePrefix --outFilterMismatchNmax 5.
To analyze the RNAPII elongation rate, we isolated the active elongation sites from paired-end bam files using bedtools genomecov (v2.29.1) with the following specifications: 5′ positions (-5), single-base resolution (-d), and strand-specificity (-strand).
Output files were converted to bedgraph format using ‘in-house’ bash script, and then to bigwig format using the UCSC tool bedGraphToBigWig (http://hgdownload.soe.ucsc.edu/admin/exe/).
Bwtool matrix v1.0 was used to cast the PRO-seq-read density on gene bodies (protein-coding) including TSSs, exon-intron junctions (excluding first and last exons), and TESs. Correct orientation was achieved by casting the forward and reverse bigwig files on coordinates of the opposite strands.
Assembly: hg19
Supplementary files format and content: big wig data of the RNA polymerase II active elongation sites in a single-base resulution
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| Submission date |
Apr 03, 2023 |
| Last update date |
Nov 07, 2023 |
| Contact name |
Gil Ast |
| E-mail(s) |
[email protected]
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| Phone |
+97236406894
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| Organization name |
Tel Aviv University
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| Street address |
Haim Levanon 55
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| City |
Tel Aviv |
| State/province |
Israel |
| ZIP/Postal code |
6997801 |
| Country |
Israel |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE228240 |
Human histone H1 variants impact splicing outcome by controlling RNA polymerase II elongation rate. |
| GSE228793 |
Human histone H1 variants impact splicing outcome by controlling RNA polymerase II elongation rate [PRO-seq] |
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| Relations |
| BioSample |
SAMN34047395 |
| SRA |
SRX19848201 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7136744_1-KO_MINUS.bw |
7.6 Gb |
(ftp)(http) |
BW |
| GSM7136744_1-KO_PLUS.bw |
7.6 Gb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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