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| Status |
Public on Feb 13, 2023 |
| Title |
SN4741_pcHiC_39_rep1 |
| Sample type |
SRA |
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| Source name |
SN4741
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| Organism |
Mus musculus |
| Characteristics |
cell line: SN4741
cell type: Neural precursor cells
genotype: WT
treatment: 39°C Incubation
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| Treatment protocol |
To induce differentiation, 24 hours after the cells were passaged, media was replaced by DMEM supplemented with 1% penicillin–streptomycin and 0.5% FBS at 39°C. Cells were allowed to grow and differentiate in these conditions for 48 hours before harvesting for experimentation.
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| Growth protocol |
SN4741 cells were maintained in high glucose Dulbecco's Modified Eagle Medium, supplemented with 1% penicillin–streptomycin and 10% fetal bovine serum in a humidified 5% CO2 incubator at 37°C. Cells at 80% confluence were passaged by trypsinization approximately every 2-3 days.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
SN4741 cells were cultured at the non-permissive temperature and plated at five million cells per 10cm dish. The cells were crosslinked using 1% formaldehyde, snap frozen using liquid nitrogen, and stored at -80°C. The cells were dounce homogenized and restriction enzyme digestion, using 400 units HindIII-HF overnight at 37°C. The total volume was maintained at 500µL, through addition of 1X NEBuffer 2.1. Heat inactivation was performed at 80°C for 20 minutes, and biotinylated-dCTP was used for biotin fill-in reaction. Blunt-end ligation was performed using Thermo T4 DNA ligase, with cohesive end units maintained at 15,000 and buffer and water volumes adjusted to ensure a total volume of 665µL was added to each Hi-C tube. Cross-linking was performed overnight, with additional (50μL) proteinase K added for two hours the following day. DNA purification was split across two reactions using 2mL PhaseLock tubes, and volumes were adjusted accordingly. Each PhaseLock reaction was split again into two vials for ethanol purification, and centrifugation at step 6.3.8 was performed at room temperature. The pellets were dissolved in 450µL 1X TLE and transferred to a 0.5mL 30kD Amicon Column. After washing, the column was inverted into a new container, and no additional liquid was added to raise the volume to 100µL. All four reactions were combined, the total volume determined, and RNAseA (1mg/mL) equal to 1% of the total volume was added for 30 minutes at 37°C.
The libraries were assessed for successful blunt-end ligation by a ClaI restriction enzyme digest of PCR products, as previously described (Belaghzal, H., Dekker, J. and Gibcus, J.H., 2017. Hi-C 2.0: An optimized Hi-C procedure for high-resolution genome-wide mapping of chromosome conformation. Methods, 123, pp.56-65.) . Biotin was removed from un-ligated ends and DNA was sheared to a size of 200-300bp using the Covaris M220 (High setting, 35 cycles of 30s “on” and 90s “off”; vortexing/spinning down samples and changing sonicator water every 5 cycles). Size selection was performed using AMpure XP magnetic beads, as described, except that all resuspension steps were increased by 5µL, so that 5μL could be used for QC with the High Sensitivity DNA Assay (Agilent) on the Agilent 2100 Bioanalyzer (at three stages: post-sonication, post-0.8x size-selection, and post-1.1x size-selection). The remaining protocol was performed as described. The remaining protocol was performed as described. Capture probes (Arbor Biosciences; https://github.com/nbbarrientos/SN4741_pcHiC) were designed against mouse (mm10) RefSeq transcription start sites, filtering out “XM” and “XR” annotated genes. The remaining promoters were intersected with the in silico digested HindIII mouse genome, to retain all HindIII fragments containing a promoter. Potential probes sites were assessed ±330bp of the HindIII cut site on either end of the fragment and finalized probe sets were filtered using no repeats and “strict” criteria, as defined by Arbor Biosciences. After generating a uniquely indexed HiC library with complete Illumina adapters, probes targeting promoter containing fragments were hybridized following Arbor Biosciences capture protocol (v4) at 65°C, 1µg DNA, and one round of capture.
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| Library strategy |
Hi-C |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
FASTQ files were mapped to mm10 using Bowtie2 (v.2.4.1) and filtered using HiCUP (v. 0.8) configured with the following parameters: FASTQ format (Format: Sanger), maximum di-tag length (Longest: 700), minimum di-tag length (shortest: 50)
BAM files were generated for each replicate using samtools (v.1.10). DeepTools (v.3.5.1) before read coverage similarities and replicate correlation was assessed using the function “multiBamSummary” (in bins mode) to analyze the entire genome.
The CHiCAGO (v. 1.18.0) pipeline was used to convert the merged BAM file into CHiCAGO format.
Assembly: mm10
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| Submission date |
Feb 10, 2023 |
| Last update date |
Feb 13, 2023 |
| Contact name |
Andrew S. McCallion |
| E-mail(s) |
andy@jhmi.edu
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| Organization name |
Johns Hopkins University School of Medicine
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| Department |
Genetic Medicine
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| Lab |
McCallion
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| Street address |
733 North Broadway
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| City |
Baltimore |
| State/province |
Maryland |
| ZIP/Postal code |
21205 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE225082 |
Evaluating the mouse neural precursor line, SN4741, as a suitable proxy for midbrain dopaminergic neurons [Hi-C] |
| GSE225084 |
Evaluating the mouse neural precursor line, SN4741, as a suitable proxy for midbrain dopaminergic neurons |
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| Relations |
| BioSample |
SAMN14064739 |
| SRA |
SRX7693037 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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