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| Status |
Public on Jul 10, 2024 |
| Title |
LB2 pA-DamID, clone SC9, parental, replicate 2, dam only |
| Sample type |
SRA |
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| Source name |
HAP1 dCAS9-KRAB cSC9
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HAP1 dCAS9-KRAB cSC9
cell type: HAP1
genotype: control
antibody: LaminB2
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| Treatment protocol |
The cell line was infected with lentiviral vectors (Lentiguide Puro: Addgene #52963) containing guide RNA for, CCSER1 (negative control). We produced lentiviral particles by transfecting HEK293T with Calcium Phosphate transfection and using pREV, pMDL, and pVSV as packaging vec-tors. Supernatants from a 10 cm dish were collected 48 hours after transfection, filtered, and concentrated to 250 µl using Amicon filters, aliquoted, and stored at -80°C. HAP1 cells were infected with 30 µl of concentrated virus and then selected for puromycin resistance 48 hours after transduction. We performed mRNA extraction 144 hours following lentiguide transduction for RNAseq
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| Growth protocol |
HAP1 cell line were grown in IMDM (Gibco, 15070063) FBS0 10%, 1% penicillin-streptomycin (Gibco, 15070063) . Cells were passaged every 2 days. Mycoplasma contamination was ruled out by regular testings (#LT07-318; Lonza).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
LMNB2 pA-DamID maps were generated as previously described (van Shaik, 2022, 36050765). Briefly, one million of HAP1 were collected by centrifugation (500 g, 3 min) and washed sequentially in ice-cold PBS and digitonin wash buffer (D-Wash) (20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.02% digitonin, cOmplete Protease Inhibitor Cocktail). Cells were rotated for 2 hours at 4 °C in 200 μL D-Wash with 1:200 LMNB2 (Abcam, ab8983) followed by a wash step with D-Wash. Following this step, cells were incubated with a solution of D-Wash buffer and Rabbit-anti-mouse IgG (1:200, Abcam, ab6709), followed by a wash step. This incubation was repeated with a 1:200 pA-Dam solution (equivalent to nearly 60 Dam units from NEB), followed by two wash steps. Dam activity was induced by incubation for 30 min at 37 °C in 100 μL D-Wash supplemented with the methyl donor SAM (30 µM) while gently shaking (500 rpm). Genomic DNA was isolated and processed similarly to DamID, except that the DpnII digestion was omitted, and 65-bp reads were sequenced. For every condition, another 1 million cells were processed in only D-Wash and, during Dam activation, incubated with 4 units of Dam enzyme (NEB, M0222L). We use Dam control samples to account for DNA accessibility and amplification biases.
Genomic DNA was isolated (Bioline, bio-52067) and ~ 500 ng were digested with DpnI (10 U, NEB, R0176L) in CutSmart Buffer 1X (8h 37°C, 20 min 80°C) in a total volume of 10 µL. A-tailing was performed by addition of 5 µL of the A-tailing mix (0.5 µL of Cutsmart buffer 10X, 0.25 µL Klenow 50 U/µL (NEB, M0212M), 0.05 µL dATP 100 mM, 4.2 µL H2O) and incubation 30 min at 37°C followed by 20 min at 75°C. Adapters were ligated by adding 15 µL of the ligation mix (3 µL T4 Ligase Buffer 10X, 0.5 µL T4 ligase (5 U/µL , Roche, 10799009001), 0.25 µl of x-Gene Stubby Adapter 50 mM (IDT), 11.25 µl H2O) and incubating samples 16h at 16°C and 10 min at 65°C. Finally, the Methyl Indexed PCR was performed by mixing 4 µL of ligated DNA with x-Gen Dual combinatorial Indexes (IDT) (125 nM final concentration) and MyTaq RedMix (Bioline, BIO-25048) in a final volume of 40 µL. The following PCR program was used: 1 X (1 min 94°C), 14 X (30 sec 94°C, 30 sec 58°C, 30 sec 72°C), 1X (2 min 72°C). Libraries were sequenced on Illumina platform Hiseq2500 with single end 65 bp reads.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
pA-DamID_187_144h_r2_Lmnb2-20kb.bw
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| Data processing |
The full protocol is described in the corresponding manuscript.
The constant sequence of the DamID adapter was removed using cutadapt 1.11 and custom scripts.
Remaining sequences were mapped to the human genome (hg38) using bwa mem 0.7.17.
Reads overlapping GATC fragments were counted in 20 kb bins and normalized to 1 million reads, after which a log2-ratio was calculated of LaminB1 over the Dam control using custom R scripts.
Assembly: hg38
Supplementary files format and content: BigWig files of log2-normalized LaminB2 pA-DamID scores for 20 kb genomic bins.
Library strategy: pA-DamID
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| Submission date |
Mar 18, 2024 |
| Last update date |
Jul 10, 2024 |
| Contact name |
Stefano Giustino Manzo |
| E-mail(s) |
stefano.manzo@unimi.it
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| Phone |
+393283622562
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| Organization name |
University of Milan
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| Department |
Biosciences
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| Street address |
Via Celoria 26, Dipartimento Bioscienze, Torre A, Piano 4
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| City |
Milano |
| State/province |
MI |
| ZIP/Postal code |
20133 |
| Country |
Italy |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE261868 |
Identification of Chromatin proteins involved in gene repression in lamina-associated domains (pA-DamID) |
| GSE261955 |
Identification of Chromatin proteins involved in gene repression in lamina-associated domains |
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| Relations |
| BioSample |
SAMN40533546 |
| SRA |
SRX23985306 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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