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| Status |
Public on Nov 23, 2022 |
| Title |
293T-RNA-seq-Hemin-80uM-6h-rep1 |
| Sample type |
SRA |
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| Source name |
293T cells RNA-seq with Hemin replicate1
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T
tag: Derived from embryonic kidney
treatment: treated with Hemin for 6 hours
antibody: none
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| Treatment protocol |
Following Hemin treatment for 2 hours or 6 hours, cells were collected.
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| Growth protocol |
HEK293T and HeLa were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, LONSERA). Drosophila S2 cells were obtained from Invitrogen and maintained in Schneider's medium. Mouse embryonic fibroblasts (MEF) cells were grown in DMEM with 10% FBS. All cells were cultured at 37°C with 5% CO 2 . Cells were confirmed to be mycoplasma negative using MycoBlue Mycoplasma Detector (Vazyme, D101-01). Live cells were quantified using a TC20 automated cell counter (Bio-Rad).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Following Hemin treatment for 6 hours, cells were collected and lysed with Trizol reagent. Total RNA was extracted according to the manufacturer’s instructions.
Biotin-PEG4-Hemin was combined with the Tn5-based CUT&Tag strategy to identify Hemin binding sites in the genome. Briefly, one million HEK293T or HeLa cells were resuspended in NE1 buffer (20 mM HEPES at pH 7.9, 10 mM KCl, 0.5 mM spermidine,0.1% Triton X-100, 20% Glycerol, 1× protease inhibitors) and incubated on ice for 10 minutes. After centrifugation at 1, 300 g for 10 minutes, nuclei were isolated and collected. These nuclei were washed once with PBS and resuspended with 100 μL of wash buffer (20 mM HEPES at pH 7.5, 150 mM KCl, 0.5 mM spermidine, 1× protease inhibitors). 10 μL pre-activated concanavalin A coated magnetic beads (Smart-Lifesciences) were added to these nuclei and incubated on a rotator for 10 minutes at room temperature. The supernatant was removed and bead-bound nuclei were resuspended in 100 μL of dig-wash buffer (0.05% digitonin, 20 mM HEPES at pH 7.5,150 mM KCl, 0.5 mM spermidine, 1× protease inhibitors) containing 2 mM EDTA.Biotin-PEG4-Hemin was added to the sample and rotated overnight at 4°C in the dark.For the control sample, biotin-PEG4-hydrazide was used to replace biotin-PEG4-Hemin. These nuclei were washed three times in 200 μL of dig-wash buffer to remove unbound biotin-PEG4-Hemin or biotin-PEG4-hydrazide. Then anti-biotin rabbit mAb was diluted 100-fold in 100 μL of dig-wash buffer with 2 mM EDTA and was added to the nuclei with one-hour rotation at room temperature in the dark. After washing once with dig-wash buffer, these nuclei were resuspended with 100 μL of dig-wash buffer with 2 mM EDTA and 1 μL of mouse anti-rabbit IgG, incubated for another one hour at room temperature in the dark, and washed three times with dig-wash buffer. A 1:250 dilution of pA-Tn5 adapter complex (~0.2 μM) was prepared in 100 μL of dig-300 buffer (0.05% digitonin, 20 mM HEPES at pH 7.5, 300 mM KCl, 0.5 mM spermidine, 1× protease inhibitors) and added to the nuclei followed by one-hour rotation at room temperature in the dark. After five washes with dig-300 buffer, the bead-bound nuclei were resuspended with 40 μL of tagmentation buffer (10 mM TAPS-NaOH at pH 8.5, 10 mM MgCl 2 , 7.5% DMF) and incubated at 37°C for one hour. To terminate the tagmentation reaction, 1.5 μL of 0.5 M EDTA, 0.5 μL of 10% SDS and 1 μL of 20 mg/mL proteinase K were added and incubated at 55°C for 1 hour and then for 15 minutes at 75°C. The DNA was extracted with Sera-Mag carboxylate-modified magnetic beads (GE Healthcare) for library preparation. 21 μL DNA was mixed with a universal i5 and a uniquely barcoded i7 primer, and amplified with NEB Q5 high- fidelity 2× master mix. The libraries were purified with 0.56-0.85× volume of Sera- Mag carboxylate-modified magnetic beads and subjected to the Bioanalyzer DNA analysis and Illumina sequencing.
ChIP-Rx was performed with 1×10 7 human cells and 1×10 6 MEF cells for spike-in normalization as described in a previous study [21]. For immunoprecipitation, sonicated chromatin was incubated with 10 μg specific antibodies and 15 μL of pre- blocked Protein A/G beads (Smart-Lifesciences). After extensive washes, the captured DNA was eluted for library preparation using the NEBNext Ultra II DNA library prep kit for Illumina before sequencing on a NovaSeq 6000.
qPRO-seq was performed as previously reported (Judd et al. 2020; Li et al. 2021).
G4-CUT&Tag and R-loop CUT&Tag were performed as previously described( Wang et al. 2021; Li et al. 2021)
RNA Sequencing (RNA-seq) Following Hemin treatment for 6 hours, cells were collected and lysed with Trizol reagent. Total RNA was extracted according to the manufacturer’s instructions. 1 μg total RNA was spike-in with 100 ng Drosophila S2 RNA and was further used for mRNA isolation with VAHTS mRNA Capture Beads (Vazyme, N401) following the manufacturer's instructions. Then library preparation was done using the NEBNext Ultra II Directional RNA Library Prep Kit, followed by sequencing on a NovaSeq 6000.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
RNA-seq
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| Data processing |
Biotin CUT&Tag Reads were aligned to the human genome (UCSC hg38) and Escherichia coli genome with Bowtie2 version 2.2.6, using parameters: --local --very-sensitive --no-mixed --no-discordant --phred33 -I 10 -X 700. The aligned reads were normalized with total reads aligned (reads per million, r.p.m.) and spiked-in E.coli reads
ChIP-Rx reads were aligned to the human genome (UCSC hg38) and mouse genome (mm10) with Bowtie2 version2 2.2.6 , using parameters: --local --very-sensitive. The resulting reads were normalized with the aligned mouse reads.
qPRO-seq reads were aligned to the human hg38 genome. The resulting reads were normalized to the total reads used for alignment (reads per million, r.p.m.) and converted to bigwig files for visualization in the UCSC genome browser. Heatmap and metagene plots were made for the indicated windows using the average coverage (r.p.m.).
G4-CUT&Tag and R-loop CUT&Tag reads were aligned to the human genome (UCSC hg38) and Escherichia coli genome with Bowtie2 version 2.2.6 [61], using parameters: --local --very-sensitive --no-mixed --no-discordant --phred33 -I 10 -X 700. The aligned reads were normalized with total reads aligned (reads per million, r.p.m.) and spiked-in E.coli reads.
RNA-seq reads were aligned to the human genome (UCSC hg38) or mouse genome (mm10) (the RNA-seq were normalized with the spiked-in Drosophila reads), respectively. Alignments were processed with HISAT2, allowing only uniquely mapping reads with up to three mismatches within the 150 bp reads. The read counts across each gene were counted with featureCounts version 2.0.0.
Assembly: Homo sapiens .hg38
Assembly: Mus musculus.mm10
Assembly: Escherichia coli K-12 substr. MG1655
Assembly: Drosophila melanogaster.dm6
Supplementary files format and content: Library normalized bigWig files were provided for all samples
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| Submission date |
Mar 15, 2022 |
| Last update date |
Nov 23, 2022 |
| Contact name |
Yin Zhinang |
| E-mail(s) |
yinzhinang@whu.edu.cn
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| Phone |
+8618163555312
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| Organization name |
Wuhan University
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| Lab |
Liang Lab
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| Street address |
Donghu road 185
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| City |
Wuhan |
| State/province |
Hubei |
| ZIP/Postal code |
430071 |
| Country |
China |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE198658 |
G-quadruplexes sense natural porphyrin metabolites for regulation of gene transcription and chromatin landscapes |
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| Relations |
| BioSample |
SAMN26674933 |
| SRA |
SRX14467519 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5954478_293T-RNA-seq-Hemin-80uM-6h-rep1.spikein.bw |
69.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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