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Sample GSM5343790 Query DataSets for GSM5343790
Status Public on May 28, 2021
Title E80 Macaque V1 IgG Go-CaRT Rep 1
Sample type SRA
 
Source name Embryonic Macaque Telencephalon
Organism Macaca mulatta
Characteristics tissue: Embryonic Macaque Telencephalon
developmental stage: Embryonic Day 80
Extracted molecule genomic DNA
Extraction protocol 75,000-100,000 acutely isolated cells from mouse, macaque, and human forebrain (or cultured 3T3 and HEK293T cells) were used for each experiment. Cells were resuspended in 500 mL ice cold Nuclei Isolation Buffer (NIB) (10 mM HEPES-KOH, Ph 7.9. 10 mM KCl, 0.1% NP40, 0.5 mM Spermidine and 1X Halt protease inhibitor cocktail) and incubated for 10 min in ice. Nuclear pellet was collected by centrifuging at 600xg for 3 min at 40C and again washed with 500 mL NIB. Nuclear pellet was collected by centrifugation at 600xg for 3 and resuspended in 100 mL of NIB. Successful nuclei isolation was confirmed by visual inspection under a phase contrast microscope. Next, 10 mL Concanavalin A lectin beads (Bio-Mag Plus) washed and resuspended in Binding Buffer (BiB) (20 mM HEPES-KOH, pH 7.9, 10 mM KCl, 1 mM CaCl2 and 1 mM MnCl2) were added to each sample and rotated for 10 min at room temperature. Nuclei bound Concanavalin A beads were pulled by placing the tubes on a magnetic stand to remove the supernatant. For LaminB GO-CaRT, anti LaminB1 antibody (Abcam# ab16048) was used and for SON GO-CaRT, anti-SON antibody (Atlas antibodies #HPA031755) was used at 1:100 dilution in Blocking Buffer (BlocB) (Wash buffer containing 2mM EDTA). Same steps were followed for histone marks using anti-H3K9me2 (Active Motif #39239, 1:100), anti-H3K9me3 (Abcam# ab8898; 1:100), anti-H3K27me3 (Cell signaling technologies #9733; 1:100). An IgG control was run in parallel for all experiments (Cell signaling technologies #2729; 1:100). To each sample 50 mL of BlocB containing primary antibody was added and incubated overnight on a rotator at 40C. Next day, samples were briefly spun and placed on magnetic stand to clear off the liquid. Samples were washed twice with 1mL Wash Buffer (WaB) (20 mM HEPES-KOH, pH 7.5, 150 mM NaCl, 0.1% BSA, 0.5 mM Spermidine and 1X Halt protease inhibitor cocktail). At the end of two washing steps, Concanavalin A bound nuclei were resuspended in 50 mL of WaB and gently mixed. To each tube, 2.5 mL of pA-MNase (diluted 1:10 in WaB from 140 mg/mL stock) kindly provided by Steven Henikoff (Fred Hutchison Cancer Research Center) was added and rotated for 1 hour at 40C. Samples were briefly spun and placed on magnetic stand to remove the liquid. Samples were washed twice with 1mL WaB. At the end of two washing steps, Concanavalin A bound nuclei were resuspended in 100 mL of WaB and gently mixed. The tubes were placed in pre-chilled metal blocks sitting in ice. While gently vortexing, 2 mL of 100 mM CaCl2 was added to initiate MNase digestion and returned to pre-chilled metal blocks. Digestion was carried out for 30 min and stopped by adding 100 mL of 2XSTOP (200 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 mg/mL RNAseA, 40 mg/mL Glycogen and 10 pg/mL of heterologous DNA) while gently vortexing. To release pA-MNase cleaved fragments, samples were incubated at 370C for 15 min. DNA was extracted using Phenol: Chloroform extraction method. We considered the possibility that the pA-MNase cleaved DNA-protein complexes might be too large to diffuse out of the nucleus and enter the “soluble” fraction. To test this, we recovered DNA from both soluble and total fractions (soluble + insoluble). LaminB profiles were highly similar (Pearson= 0.94) between soluble and total fractions, indicating that DNA-protein complexes released by LaminB GO-CaRT can readily diffuse out of the nucleus.
Sequencing libraries were prepared from GO-CaRT DNA fragments using KAPA HyperPlus with amplification DNA library preparation kit (#KK8512) following manufacturer instructions. The library was amplified for 13-15 PCR cycles and larger DNA fragments were depleted by doing a 0.55X size selection using Agencourt AMPure XP beads (Beckman #A63881).
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina HiSeq 4000
 
Data processing Library strategy: GO-CaRT
150bp paired-end sequencing of GO-CaRT libraries was performed on the Illumina HiSeq 4000. Low quality reads and adaptors were removed with BBduk (http://jgi.doe.gov/data-and-tools/bb-tools/), and resulting reads were aligned to the mouse (UCSC mm10), macaque (USCS rheMac8) or human (UCSC hg38) genome using bowtie2 and the following settings: --local --very-sensitive-local --no-unal --no-mixed --no-discordant -q --phred33 -I 10 -X 700. Duplicate reads were removed with Picard tools
 
Submission date May 27, 2021
Last update date May 28, 2021
Contact name Tomasz Nowakowski
E-mail(s) ryanndelgado@gmail.com
Organization name UCSF
Street address 1550 4th St
City San Francisco
State/province Ca
ZIP/Postal code 94158
Country USA
 
Platform ID GPL23949
Series (1)
GSE175679 Distinct nuclear compartment-associated genome architecture in the developing mammalian brain
Relations
BioSample SAMN19369806
SRA SRX11006816

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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