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Sample GSM3904484 Query DataSets for GSM3904484
Status Public on Jun 28, 2019
Title DamID, Dam, RPE, CRISPRa, control, rep2
Sample type SRA
 
Source name DamID, Dam, RPE, CRISPRa, control
Organism Homo sapiens
Characteristics cell line: RPE-1
genotype/cell type: RPE-1 cell line stably expressing SunTag-CRISPRa
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.
Growth protocol Cells were cultured according to 4DNucleome SOP, https://www.4dnucleome.org/cell-lines.html
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.
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina HiSeq 2500
 
Description 4591_10_RPE_sg_Dam_r2_AGTACAAG_S11
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
 
Submission date Jun 25, 2019
Last update date Jun 29, 2019
Contact name Bas van Steensel
E-mail(s) b.v.steensel@nki.nl
Phone + 31 20 512 2040
Fax +31 20 669 1383
URL http://www.nki.nl/nkidep/vansteensel
Organization name Netherlands Cancer Institute
Department division of Molecular Biology
Lab van Steensel group
Street address Plesmanlaan 121
City Amsterdam
ZIP/Postal code 1066 CX
Country Netherlands
 
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
Relations
BioSample SAMN12136100
SRA SRX6365984

Supplementary file Size Download File type/resource
GSM3904484_RPE_sg_Dam_r2-gatc.counts.txt.gz 45.3 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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