GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM7993395

Query DataSets for GSM7993395

Status

Public on Dec 29, 2023

Title

WT_ESC_Asb18_rep2

Sample type

SRA

Source name

WT mESC

Organism

Mus musculus

Characteristics

cell line: WT mESC
cell type: mESC
genotype: WT
treatment: Double hybridization capture using ssDNA probes within the Asb18 promoter (viewpoint coordinates_ chr1_90013013-90013385)

Treatment protocol

Transgenic mESC lines with different genomic re-arrangements were generated using CRISPR/Cas9 technology. The design of the CRISPR/Cas9 guide sequences (gRNA) was performed using the CRISPR Benchling software tool (https_//www.benchling.com/crispr/) (Data S3). In order to increase the cutting efficiency of the gRNA, a guanine nucleotide was added at the first position of the sequence and a restriction site for the BbsI enzyme (R0539L, NEB) was added to the beginning of the gRNA for cloning purposes. For each sgRNA, two oligonucleotides were synthesized (Sigma) annealed and cloned into a CRISPR-Cas9 expression vector (pX330-hCas9-long-chimeric-grna-g2p; gift from Leo Kurian’s laboratory). The hybridized oligos were cloned into the pX330-hCas9-long-chimeric-grna-g2p using 50 ng of BbsI-digested vectors and 1 ul ligase (Thermo Fisher, EL0013) in a total volume of 20 μl. The ligation reaction was incubated for 1 h at room temperature. For each cell line, ESC were transfected with the corresponding pair of gRNAs-Cas9 expressing vectors using Lipofectamine following manufactured recommendations (Thermo Scientific, L3000001). After 24 hours, transfected cells were selected by treating them with for 48 hours. Single-cell isolation of surviving cells was performed by serial dilution and seeding in 96-well plates. Next, clones with the desired genetic rearrangements (i.e. deletions or inversions) were identified by PCR using the primers listed in Data S3. DNA extraction was performed using Lysis Buffer_ 25 mM KCl (SigmaAldrich, 27810.295), 5mM TRIS (Sigma-Aldrich, 0497-5KG) pH8.3, 1.25mM MgCl2 (VWR BDH7899-1), 0.225% IGEPAL (Sigma-Aldrich, I8896-50ML) and 0.225% Tween20 (VWR). Proteinase K (ThermoFisher Scientific, EO0492) was added to a final concentration of 0.4 µg/ul before use.

Growth protocol

E14Tg2a (E14) mouse ESCs were cultured on gelatin-coated plates using Knock-out DMEM (Life Technologies, 10829018) supplemented with 15% FBS (Life Technologies, 10082147), leukemia inhibitory factor (LIF), antifungal and antibiotics (Sigma-Aldrich, A5955), β-mercaptoethanol (ThermoFisher Scientific, 21985023), Glutamax (ThermoFisher Scientific, 35050038) and MEM NEAA (ThermoFisher Scientific, 11140035). Cells were cultured at 37°C with 5% C02. For the NPC differentiation 80, ESCs were plated at 20,000 cells/cm2 on geltrex-coated plates (ThermoFisher, A1413302) and grown in N2B27 medium_ Advanced Dulbecco’s Modified Eagle Medium F12 (Life Technologies, 21041025) and Neurobasal medium (Life Technologies, 12348017) (1_1), supplemented with 1× N2 (R&D Systems, AR009), 1× B27 (Life Technologies, 12587010), 2 mM L-glutamine (Life Technologies, 25030024) and 0.1 mM 2-mercaptoethanol (Life Technologies, 31350010)). During the six days of differentiation, the N2B27 medium was additionally supplemented with the following components_ bFGF (ThermoFisher Scientific, PHG0368) 10 ng/ml from day 0 to day 2, Xav939 (Sigma-Aldrich, X3004-5MG) 5µM from day 2 to day 6, BSA (ThermoFisher Scientific, 15260037) 1mg/ml at day 0 and 40 µg/mL the remaining days.

Extracted molecule

genomic DNA

Extraction protocol

Capture-C experiments were performed as previously described. 5 _ 106 cells were crosslinked with 2% formaldehyde for 10 minutes and quenched with 0.125 M glycine for 10 minutes. Cells were washed with PBS and resuspended in lysis buffer (10 mM Tris pH 8, 10 mM NaCl, 0.2% NP-40 and 1_ protease inhibitors) during 20 minutes on ice. Following centrifugation, the pellet was resuspended in 215 µL 1_CutSmart buffer and transferred to a microcentrifuge tube. The resuspended pellet was mixed with 60 ul 10_ CutSmart buffer, 393,5 µl water and 9,5 µl 20% (vol/vol) SDS (0.28% final concentration) (Invitrogen, cat. no. AM9820) followed by 1 h incubation at 37¡C while shaking on a thermomixer at 500 rpm (intermittent shaking_ 30s on / 30s off). Then, 20% vol/vol Triton X 100 was added at a final concentration of 1.67% vol/vol followed by another incubation at 37¡C for 1h while shaking. Chromatin was digested by adding 25 µL NlaIII (250 U, R0125L) and incubating at 37 ¡C for several hours, followed by addition of another 25 µL of NlaIII and incubation at 37 ¡C overnight. The digested chromatin was ligated with 8 µl (240U) of T4 DNA ligase (Life Tech, cat. no. EL0013) for 18 hours at 16¡C. Samples were treated with Proteinase K (3U; Thermo Fisher, cat. no. EO0491) and RNase A (7,5 mU; Roche_ 1119915), and DNA was purified using the Qiagen kit (28506). After checking the quality of the digestion and subsequent ligation, chromatin was sonicated for 30 cycles (30 s on, 30 s off, 25% amplitude) using an EpiShear probe sonicator (Active Motif) and DNA samples were purified using AMPure XP SPRI beads (Beckman Coulter, cat. no. A63881).
Libraries were prepared using the NEBNext Ultra II kit (New England Biolabs, cat. no. E7645S/L). Index primers set 1 and 2 from the NEBNext Multiplex Oligos for Illumina kit (New England Biolabs, E7335S/L E7500S/L) were incorporated using Herculase II Fusion Polymerase Kit (Agilent, cat. no. 600677). The resulting libraries were pooled (six libraries/pool) and a double Hybridization capture using ssDNA probes was performed following a modified version of the Roche HyperCapture streptavidin pull-down protocol (Roche, cat. no. 09075763001) described in 81. Libraries were sequenced using Novaseq6000_150PE_2,25Gb/lib (15 Mreads/lib). For each of the investigated cell lines, Capture-C experiments were performed as two independent biological replicates.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina NovaSeq 6000

Data processing

Capture C reads were subject to quality control and trimming of low quality regions and/or adapters using fastqc (https_//www.bioinformatics.babraham.ac.uk/projects/fastqc/), MultiQC and trimmomatic .
Reads were processed with capC-MAP, considering the restriction enzyme NlaIII (cutting site CATG), the mm10 reference genome and normalize = TRUE. The coordinates (mm10) of the viewpoints (i.e. «targets» according to capC-MAP terminology) were_ Gbx2 SE _ chr1 89869929 89870764 ; Six3 SE _ chr17 85484699 85485038; Asb18 TSS_ chr1 90013014 90013383; Six2 TSS_ chr17 85688402 85689017
After running capC-MAP, the normalized pileup bedgraph files for intra-chromosomal contacts for each viewpoint were collected. According to capC-MAP documentation, the number of piled-up interactions per restriction fragment are normalized to reads per million, so that the sum ofthe number of reads associated to each viewpoint genome-wide is equal to one million. Subsequently, for the restriction sites without any detected interactions, a signal equal to 0 wasassigned. In addition, only data from the regions chr1 89228752 90664659 (Gbx2-Asb18 locus) and chr17 84890284 86289254 (Six3-Six2 locus) was considered. Next, the bedgraph files of both replicates were averaged and the resulting bedgraph was converted to a bigwig with the usage of thebedGraphToBigWig UCSC tool.
Assembly: mm10
Supplementary files format and content: Bigwig; for each sample the bigwig files show the average Capture-C signals obtained for two independent biological replicates (rep1 and rep2)

Submission date

Dec 26, 2023

Last update date

Dec 29, 2023

Contact name

Alvaro Rada-Iglesias

E-mail(s)

alvaro.rada@unican.es

Organization name

Institute of Biomedicine and Biotechnology of Cantabria (IBBTEC), CSIC/University of Cantabria

Street address

Albert Einstein 22, PCTCAN