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| Status |
Public on Jun 28, 2019 |
| Title |
DamID, Dam-LMNB1, RPE, CRISPRa, TRAM1L1, rep1 |
| Sample type |
SRA |
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| Source name |
DamID, Dam-LMNB1, RPE, CRISPRa, TRAM1L1
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| Organism |
Homo sapiens |
| Characteristics |
cell line: RPE-1
genotype/cell type: RPE-1 cell line stably expressing SunTag-CRISPRa
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| Treatment protocol |
RPE-1 cells: sgRNAs were cloned into Lentiguide-Puro vector (Addgene #52963) using restriction enzyme BsmBI, and Lentivirus was prepared. RPE-1 cells stably expressing SunTag-CRISPRa were infected with LentiGuide virus and selected with 10 µg/µl Puromycin for one week to obtain stable polyclonal cell pools.
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| Growth protocol |
Cells were cultured according to 4DNucleome SOP, https://www.4dnucleome.org/cell-lines.html
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
sgRNAs were cloned into Lentiguide-Puro vector (Addgene#52963) using restriction enzyme BsmBI and Lentivirus was prepared. CRISPRa RPE cells were infected with LentiGuide virus and selected with 10 µg/µl Puromycin for one week to obtain stable polyclonal cell lines. DamID-seq was performed as described in (Brueckner et al. 2016) with slight modifications. Instead of plasmid transfections, Dam-LMNB1 or Dam were added by Lentiviral transduction. Three days after infection, cells were collected for gDNA isolation. gDNA was pre-treated with SAP (10 U, New England Biolabs) in CutSmart buffer in a total volume of 10 µl at 37 °C for 1 h followed by heat inactivation at 65°C for 20 min to remove signal from apoptotic fragments. gDNA was digested with DpnI (10 U, New England Biolabs) in CutSmart buffer in a total volume of 10 µl at 37 °C for 8 h followed by heat inactivation at 80 °C for 20 min. Fragments were ligated to 12.5 pmol DamID adapters using T4 ligase (2.5 U, New England Biolabs) in T4 ligase buffer in a total volume of 20 µl incubated at 16 °C for 16 h. The reaction was heat inactivated for 10 min at 65 °C. Products were then digested with DpnII to remove partially methylated fragments. DpnII buffer and DpnII (10 U, New England Biolabs) were added in a total volume of 50 µl and incubated at 37 °C for 1 h. 8 µl of DpnII-digested products was amplified by PCR with MyTaq Red Mix (Bioline) and 2.5 µM primers Adr-PCR-Rand1 in a total volume of 40 µl. PCR settings were 8 min at 72 °C (1×) followed by 20 s at 94 °C, 30 s at 58 °C, 20 s at 72 °C (24× for Dam, 28x for DamLMNB1 samples) and 2 min at 72 °C (1×). Remaining steps were performed as previously described.
DamID-seq was performed as described in (Brueckner et al. 2016) with slight modifications. Instead of plasmid transfections, Dam-LMNB1 or Dam were added by Lentiviral transduction. Three days after infection, cells were collected for gDNA isolation. gDNA was pre-treated with SAP (10 U, New England Biolabs) in CutSmart buffer in a total volume of 10 µl at 37 °C for 1 h followed by heat inactivation at 65°C for 20 min to remove signal from apoptotic fragments. gDNA was digested with DpnI (10 U, New England Biolabs) in CutSmart buffer in a total volume of 10 µl at 37 °C for 8 h followed by heat inactivation at 80 °C for 20 min. Fragments were ligated to 12.5 pmol DamID adapters using T4 ligase (2.5 U, New England Biolabs) in T4 ligase buffer in a total volume of 20 µl incubated at 16 °C for 16 h. The reaction was heat inactivated for 10 min at 65 °C. Products were then digested with DpnII to remove partially methylated fragments. DpnII buffer and DpnII (10 U, New England Biolabs) were added in a total volume of 50 µl and incubated at 37 °C for 1 h. 8 µl of DpnII-digested products was amplified by PCR with MyTaq Red Mix (Bioline) and 2.5 µM primers Adr-PCR-Rand1 in a total volume of 40 µl. PCR settings were 8 min at 72 °C (1×) followed by 20 s at 94 °C, 30 s at 58 °C, 20 s at 72 °C (24× for Dam, 28x for DamLMNB1 samples) and 2 min at 72 °C (1×). Remaining steps were performed as previously described.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
4591_24_RPE_164_DamLAM_AGAGTCAA_S24
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| Data processing |
Data analysis of DamID samples: First, the constant DamID adapter was trimmed from the 65 bp single-end reads (with cutadapt 1.11 and custom scripts). The remaining gDNA starting with GATC was mapped to hg19 with bowtie2 2.2.6. Uniquely mapped reads (filtered for bowtie’s XS-tag) were counted to GATC fragments.
Genome_build: hg19 for homo sapiens samples, mm10 for mus musculus samples
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| Submission date |
Jun 25, 2019 |
| Last update date |
Jun 29, 2019 |
| Contact name |
Bas van Steensel |
| E-mail(s) |
b.v.steensel@nki.nl
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| Phone |
+ 31 20 512 2040
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| Fax |
+31 20 669 1383
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| URL |
http://www.nki.nl/nkidep/vansteensel
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| Organization name |
Netherlands Cancer Institute
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| Department |
division of Molecular Biology
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| Lab |
van Steensel group
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| Street address |
Plesmanlaan 121
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| City |
Amsterdam |
| ZIP/Postal code |
1066 CX |
| Country |
Netherlands |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE133272 |
Effects of transcription on genome - nuclear lamina interactions: DamID-seq data |
| GSE133275 |
Effects of transcription on genome - nuclear lamina interactions |
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| Relations |
| BioSample |
SAMN12136120 |
| SRA |
SRX6366070 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3904569_RPE_164_DamLAM_r1-gatc.counts.txt.gz |
46.0 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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