GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM5343736

Query DataSets for GSM5343736

Status

Public on May 28, 2021

Title

HEK293T IgG GO-CaRT Rep2

Sample type

SRA

Source name

HEK293T

Organism

Homo sapiens

Characteristics

tissue: HEK293T

Growth protocol

Cells were cultured in DMEM with 10% FBS

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