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| Status |
Public on Oct 19, 2023 |
| Title |
MouseNeuronCulturePositiveSingle, rep21 |
| Sample type |
SRA |
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| Source name |
brain
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| Organism |
Mus musculus |
| Characteristics |
tissue: brain
cell line: N/A
cell type: neuron
genotype: C57/BL6
probe id: 502
primer 2p: 505b
primer pc: 302
has probe: Y
has laser405: Y
has laser633: Y
cell count: 1
harvest method: culture
has tpa: N
tpa dose: none
tpa time: none
has mung_bean: N
is negative_control: N
composition: Single
biological group: MouseNeuronCulture
treatment group: PositiveSingle
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| Treatment protocol |
The cultured cells were transferred to a 50 ml tube and 16% paraformaldehyde (final 1%) was added for 10 mins at room temperature to fix the cells. After fixation, 1 M glycine (final 200 mM) with RPMI 1640 medium was used to quench for 10 mins followed by centrifugation at 300 x g for 5 mins. The supernatant was discarded and - 3 mL of PBS were added to the pellet and then mixed by gently pipetting up and down 10-15 times using a fire-polished glass-pipette, to prevent cell clumping, and centrifuged at 300 rpm for 5 mins. The 100 μl cell pellet was attached to 18 mm gridded coverslips by incubating them for 2 hr. at room temperature. The samples were treated with PBS (w/o Ca++, Mg++) containing 0.01% Triton X-100 for 10 mins and then washed with PBS (w/o Ca++, Mg++) 3 times for 3 mins. To prepare human neuronal cell cultures, adult human brain tissue was placed in the papain (20 U, Worthington Biochemical) solution to dissociate at 37 °C for 30 to 40 mins and followed by ovomucoid (a papain inhibitor, 10 mg/ml, Worthington Biochemical) to stop the enzymatic dissociation. The tissue was triturated with a fire-polished glass Pasteur pipette. The cloudy cell suspension was carefully transferred to a new tube and centrifuged at 300 x g for 5 mins at room temperature. The cells were counted in an Autocounter (Invitrogen). Cells were plated on poly-L-lysine-coated (0.1 mg/ml, Sigma-Aldrich) 12-mm coverslips at a density of 3 × 104 cells/coverslip. Cultures were incubated at 37 °C, 95% humidity, and 5% CO2 in neuronal basal medium (Neurobasal A, Gibco), serum-free supplement (B-27, Gibco) and 1% penicillin/streptomycin (Thermo-Fisher Scientific). Dispersed mouse neuron/astrocyte cultures were prepared following published protocols. Dispersed cells were fixed using 4% paraformaldehyde for 10 min at room temperature. This was followed by three washes with 1x PBS. The cells were permeabolized with 0.1% Triton-X100 for 10 min at room temperature followed by three washes with 1x PBS.
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| Growth protocol |
K562 cells were obtained from ATCC and cultured in RPMI 1640 medium (Invitrogen) with 10% FBS and penicillin-streptomycin in a T75 flask at 37 °C in 5% CO2 for 2~3 days. HumanInterneuronCulture samples were derived from human induced pluripotent stem cells (iPSCs), where Stewart-4wk-human stem cells from the Children's Hospital of Philadelphia (CHOP) were plated on rat cortical neuronal feeders-1 and selected by interneuron promoter Lhx6-driven GFP expression. For mouse samples, a 3-month old male mouse was anaesthetized with halothane, euthanized by thoracotomy, then subjected to cardiac perfusion with 5 ml PBS followed by 20 ml PBS/4% paraformaldehyde. The brain was removed and post fixed at 4 °C for 16 hr., then rinsed in PBS and sectioned in the coronal plane at 100 μm on a vibratome (Leica VT-1000s). Sections including the hippocampus were then subjected to immunofluorescence labeling with chicken anti-MAP2 antisera (1:1000; Ab 5392; Abcam) followed by Alexa 488 conjugated goat anti-chicken secondary antibody (1:400; ab150169; Abcam).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
After fixation and permeabilization, the cells and brain slices were incubated with CHEX-seq probe (170 nM) in TES buffer (10 mM Tris, 1 mM EDTA, 150 mM NaCl) for 1 hr at room temperature. The samples were then washed with 1x PBS (w/o Ca++, Mg++) 3 times for 3 min. After CHEX-seq probe annealing and washing, the samples were transferred to the imaging chamber with 1x PBS (w/o Ca++, Mg++). All images and photoactivations were performed using a Carl Zeiss 710 Meta confocal microscope (20x water-immersion objectives, NA 1.0). CHEX-seq probe annealing was confirmed by exciting at 633 nm and emission was detected at 640-747 nm. The photoactivation was performed using the 405 nm (UV) laser at 60% power and 6.30 μs per pixel. After photoactivation in each individual cell’s nucleus, a master mix containing DNA polymerase I and 1st strand DNA synthesis buffer was added to the cells and incubated for 1 h at room temperature. Subsequently, the single cells containing synthesized complementary DNA were harvested using a glass micropipette under using a Zeiss 710 confocal microscope (Carl Zeiss) for visualization.
(A) 1st strand DNA synthesis and poly G tailing at 3’ end: After harvesting single cells, the in situ synthesized cDNA was removed by adding fresh prepared 0.1 N NaOH and incubating the sample for 5 min at RT followed by neutralization with 1 M Tris (pH 7.5). After ethanol precipitation, the 1st strand DNA was resuspended in nuclease free water. Subsequently, poly(G) was added to the 3’ end using terminal deoxynucleotidyl transferase (TdT) (Invitrogen). (B) 2nd strand DNA synthesis and round 1 linear RNA amplification: 2nd strand DNA was synthesized using DNA polymerase I for 2 h at 16 °C after priming with custom App-RC-polyC primer (Supplemental Data, Table 1). RNA was amplified using linear in vitro transcription from T7 RNA polymerase promoter incorporated into the double-stranded DNA with Ambion MEGAscript T7 In Vitro Transcription (IVT) Kit. (C) Round 2 1st and 2nd strand DNA synthesis and PCR amplification: After cleanup IVT reaction, 1st strand DNA was reverse transcribed from aRNA using Superscript III using a custom App-RC primer (Supplemental Data, Table 1) 2nd strand DNA was synthesized using DNA Polymerase 1 with a custom 18bpPBC1 primer (Supplemental Data, Table 1). Subsequently, the double-stranded blunt ended DNA was amplified using custom primers 18bpPBC1 / App-RC (Supplemental Data, Table 1) following PCR condition: 98 °C for 30 sec; thermocycling at 98 °C for 10 sec, 50 °C for 30 sec, 72 °C for 30 sec for 27 cycles; extension at 72 °C for 2 mins, and was then used for library construction. Samples for the control experiments were processed with the same procedure except no CHEX-seq probe was applied, and 2nd round 2nd strand DNA PCR amplification was performed with custom primers 18bpPBC14 / App-RC. Illumina TruSeq Nano DNA Library Preparation Kit was used with modifications. All of the second round PCR amplified double-stranded DNA was used as input. After converting DNA fragment into blunt ends with End Repair Mix, base “A” was added; sequence adapters were ligated. DNA inserts were amplified with PCR.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
MouseNeuronCulturePositiveSingle
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| Data processing |
Base calls and demultiplexed using bcl2fastq v2.17. Reads were first preprocessed by a customized SCAP-T Next Generation Sequencing pipeline (https://github.com/safisher/ngs), then mapped back to the Gencode GRCh38 (human) or UCSC mm10 (mouse) genome. Further, an additional QC procedure was applied to filter for high-quality reads with respect to the barcode/primer quality as well as the alignment quality. Basically, reads that qualify the A, B and C barcode/primer class are retained. Afterwards, reads that are less than 20bp (non-redundant length if paired) or have more than 10% mismatches are filtered out. Finally, reads that are mapped to low-quality genomic regions (defined by synthetic single-end 20bp reads that are mis-aligned to respective genomes) are removed. In addition, the ENCODE listed blacklist (https://sites.google.com/site/anshulkundaje/projects/blacklists) for human or mouse was used to mask the promiscuous regions. Finally, the starts (i.e. 5' end) of priming sites were called and output in the BED format, which were used for priming frequency estimation.
For priming start sites, we used R package ChIPseeker (v1.20.0) to annnote their genomic location with respect to promoter proximal as well as distal regions. To estimate the priming frequency, we used R package GenomicRanges v1.34.0. For priming frequency estimation, we used an in-house procedure to generate the aggreated coverage profiles at various genomic features, including TSS, gene body, intergenic, exon, intron, CDS. The genome annotation used for human was Ensembl v86 (via R package EnsDb.Hsapiens.v86), and for mouse was Ensembl v79 (via R package EnsDb.Mmusculus.v79).
Assembly: hg38, mm10
Supplementary files format and content: BED files containing the 5' location of CHEX-seq probe priming
Library strategy: CHEX-seq
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| Submission date |
May 04, 2023 |
| Last update date |
May 02, 2024 |
| Contact name |
Junhyong Kim |
| Organization name |
University of Pennsylvania
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| Department |
Biology
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| Lab |
Junhyong Kim
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| Street address |
433 S University Avenue
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE231719 |
Light-Assisted Single-Stranded DNA Open-Chromatin Analysis in Single Spatially Localized Neuronal Cells Identifies the Capacity of Endogenous DNA to Act Catalytically [CHEX-seq] |
| GSE232216 |
Light-Assisted Single-Stranded Open-Chromatin Analysis in K562 and Spatially Localized Neuronal Single Cells |
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| Relations |
| BioSample |
SAMN34591058 |
| SRA |
SRX20228717 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7299036_scCLTdegenNuc526.5end.bed.gz |
6.9 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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