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| Status |
Public on Jun 16, 2021 |
| Title |
Pol2-ser2 |
| Sample type |
SRA |
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| Source name |
HEK293
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293
antibody: Pol2-ser2
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| Growth protocol |
Cells were cultured in complete DMEM medium (Sigma), 10% fetal bovine serum (Sigma), 2 mg/ml L-alanyl-Lglutamine (Biological Industries Israel), 100 U/ml penicillin, 0.1 mg/ml streptomycin (Biological Industries Israel) at 37 °C in a humidified atmosphere with 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Approximately 10×106 cells per sample were trypsinized, washed with PBS, and crosslinked with 1% FA at 37 °C. After incubation for 10 min, the reaction was stopped by addition of glycine (0.125 M) and incubated at 37 °C for 5 min. Samples were centrifuged at 2300 x g, washed with cold PBS, and centrifuged again. Nuclei isolation, MNase (Worthington) digestion, and IP were done as described (Kfir et al. 2015) with the following minor adaptations: After suspension in IP buffer, the samples were sonicated using a Bioruptor (Diagenode) at 40% amplitude in intervals of 1.5-sec pulses with 9.9-sec pauses for 10 min.
For the double ChIP-seq, ChIP was performed as described previously (Yearim et al. 2015) with the following adaptations: Approximately 7×106 HEK293 cells were used per sample. After nuclei purification and MNase digestion, samples were sonicated using a Bioruptor at 40% amplitude with intervals of 2.2-sec pulses with 9.9-sec pauses for 12 min. For anti-pol II p-Ser2 IP, 80 µl of a mixture of protein A and G Dynabeads (Invitrogen) were used with 18 µg anti-pol II p-Ser2 antibody (Abcam; ab5095). After IP, 1 µl of 10 mg/ml RNase A (Sigma) was added, and samples were incubated for 30 min at 37 °C. Following washes, the samples were eluted with 50 μl fresh 0.1 M DTT. As previously described (Sadeh et al. 2016), 50 µl of freshly prepared 2X Chromatin Release Buffer (500 mM NaCl, 2% deoxycholate, 2% SDS, 2 mM EDTA) with fresh EDTAfree protease inhibitor cocktail and PMSF (from the RNA ChIP-IT kit) were added, and samples were incubated at 37 °C for 55 min. The elution step was repeated and samples were incubated for 30 min at a temperature higher than 15 °C to prevent SDS precipitation. This step releases bound chromatin and inactivates the antibodies used in the first ChIP. To the eluted samples 1.5 µl Proteinase K (NEB) was added, and samples were incubated for 16 h at 65 ºC. DNA was purified using phenol:chloroform:isoamyl alcohol (Sigma) extraction. For the second ChIP, 50 µl of a mixture of protein A and G Dynabeads (Invitrogen) were used with 10 µg anti-U1C antibody (Abcam; ab157116). After incubation for 16 h at 4 ºC, the samples were washed and eluted as described previously (Yearim et al. 2015).
Deep sequencing libraries were prepared using Illumina TruSeq library preparation kits as per the manufacturer’s instructions. Sequencing of 50-bp single-end reads was performed using an Illumina HiSeq 2000.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Raw reads that passed the quality controls were aligned to human genome assembly hg19 using bowtie2 (Langmead and Salzberg , 2012) with parameter -very-sensitive
Only uniquely aligned reads were kept and duplicated reads were removed
multiple reads that were mapped to the same location in the genome with the same UMI in the same sample were removed
Sequencing depth files at single-base resolution were created using bam2wig.pl tool (http://search.cpan.org/~tjparnell/Bio-ToolBox-1.44) and for each base a normalized reads-per-million value was calculated and assigned considering all the reads that span that base.
The normalized input coverage was subtracted from the normalized ChIP-seq and RNA-Seq coverage to represent the difference between the two samples and the final occupancy value for each base.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include normalized RPM values for each sample
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| Submission date |
Feb 06, 2020 |
| Last update date |
Jun 16, 2021 |
| Contact name |
Matan Sorek |
| E-mail(s) |
matan.sorek@mail.huji.ac.il
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| Phone |
972-2-6585144
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| Organization name |
The Hebrew University of Jerusalem
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| Department |
Genetics
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| Lab |
Prof. Eran Meshorer
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| Street address |
Edmond J. Safra Campus
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| City |
Jerusalem |
| ZIP/Postal code |
91904 |
| Country |
Israel |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE144874 |
Intron looping is mediated by U1 snRNP and RNA polymerase II co-transcriptionally [ChIP-seq] |
| GSE145092 |
Intron looping is mediated by U1 snRNP and RNA polymerase II co-transcriptionally |
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| Relations |
| BioSample |
SAMN14051814 |
| SRA |
SRX7691494 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4299826_pol2_ser2.bw |
532.0 Mb |
(ftp)(http) |
BW |
| GSM4299826_pol2_ser2_normalized.bw |
2.5 Gb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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