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Status |
Public on May 04, 2020 |
Title |
RIC-seq_pcp_minus_HeLa_Total_rep1 |
Sample type |
SRA |
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Source name |
human HeLa cells
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Organism |
Homo sapiens |
Characteristics |
cell line: HeLa
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Treatment protocol |
Cells were fixed in 10 ml of PBS containing 1% formaldehyde at room temperature for 10 min and then quenched by addition of 1/20 volume of 2.5 M glycine. After washing three times with ice-cold PBS, cells were scraped into 15 ml tubes and collected by centrifugation at 2,500 rpm for 10 min at 4 °C. The cell pellet was resuspended in 1 ml of permeabilization buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.5% NP-40, 0.3% Triton X-100, 0.1% Tween 20, 1× protease inhibitors, 2 U/ml SUPERase In RNase inhibitor), mixed briefly and incubated on ice for 15 min. The integrity of the cells was examined at this step under a microscope. Cells were spun down at 3,500 rpm for 5 min at 4 °C. After washing three times with 1 × PNK buffer (50 mM Tris-HCl pH7.4, 10 mM MgCl2, 0.2% NP-40), the cell pellet was briefly incubated with 200 μl of 1 × MN mixture (50 mM Tris-HCl pH 8.0, 5 mM CaCl2, 0.03 U/μl micrococcal nuclease) for 10 min at 37 °C. The reaction was stopped by washing twice with 1 × PNK + EGTA buffer (50 mM Tris- HCl pH7.4, 20 mM EGTA 0.5% NP-40) and twice with 1 × PNK buffer (50 mM Tris-HCl pH7.4, 10 mM MgCl2, 0.2% NP-40).
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Growth protocol |
HeLa and 293T cells were grown in DMEM containing 10% fetal bovine serum plus 100 U/ml penicillin/streptomycin (Life Technologies; 15140) at 37 °C in 5% CO2. HT29 and HCT116 cells were cultured in modified McCoy’s 5A supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin. GM12878 cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin. MCF-7 cells were maintained in MEM containing 10% fetal bovine serum and 100 U/ml penicillin/streptomycin. IMR-90 cells were grown in DMEM/F12 + Glutamax (Life Technologies; 10565) supplemented with 5 ng/ml fibroblast growth factor-basic (R&D), 1 μg/ml hydrocortisone hemisuccinate, 50 μg/ml ascorbic acid, 5 μg/ml rh Insulin, 2% fetal bovine serum and 100 U/ml penicillin/streptomycin. S2 cells were maintained in Schneider’s drosophila medium supplemented with 10% heat-inactivated fetal bovine serum and 100 U/ml penicillin/streptomycin.
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Extracted molecule |
total RNA |
Extraction protocol |
After pCp-biotin labeling and proximity ligation, 200 μl of proteinase K buffer (10 mM Tris-HCl pH 7.5, 10 mM EDTA, 0.5% SDS) and 50 μl of proteinase K were applied to cell pellets and sequentially incubated at 37 °C for 60 min and 56 °C for 15 min. The RNA was extracted with 750 μl of TRIzol LS and 220 μl of chloroform following the manufacture’s instruction. To remove the potential RNA-DNA ligation byproducts mediated by T4 RNA ligase, 20 μg of total RNA was treated with a mixture containing 10 μl of 10 × RQ1 DNase I buffer, 3 μl of RNAsin and 5 μl of DNase I at 37 °C for 20 min. RNA was then purified by acid Phenol: chloroform extraction and ethanol precipitation. The rRNA can be optionally removed using the Ribo-off rRNA Depletion Kit (Vazyme, N406-01) or one can directly proceed to the next step (to probe the rRNA structure and interactions). For fragmentation, 16 μl of total RNA was mixed with 4 μl of 5 × first-strand buffer (250 mM Tris-HCl pH 8.3, 375 mM KCl, 15 mM MgCl2) and incubated at 94 °C for 5 min, and then quickly put on ice. To enrich pCp-biotin marked chimeric RNAs, 20 μl of pre-blocked MyOne Streptavidin C1 beads, 30 μl of NF-H2O and 50 μl of 2 × TWB buffer were applied to each RNA sample and incubated at room temperature for 30 min by rotating. After washing four times with 1× TWB buffer, the beads were resuspended in 100 μl of PK buffer (100 mM NaCl, 10 mM Tris-HCl pH 7.0, 1 mM EDTA, 0.5% SDS) and incubated in Thermomixer at 95 °C for 10 min (1000 rpm). The tube was quickly placed on a magnet stand for 1 min and the supernatant was transferred to a new EP tube. This elution process was repeated two more times. Finally, the enriched RNAs were extracted with acid Phenol: chloroform and precipitated at the final concentration of 300 mM NaCl, with 3 volumes of -20°C ethanol and 1 μl of glycoblue. RNA was pelleted at 13,000 rpm and washed twice with 75% ETOH before resuspending in 10 μl of nuclease-free water. To synthesis first-strand cDNA, 0.5 μl of N6 primer (0.1 μg/μl) was added to the mixture and incubated at 65 °C for 5 min, followed by rapid placement on ice for at least 2 min. Reverse transcription was performed by further addition of 5.5 μl of reverse transcription mix (3 μl of 5 × first-strand buffer, 1 μl of 10 mM dNTPs, 0.5 μl of 100 mM DTT, 0.5 μl of RNase inhibitor, 0.5 μl of Superscript II reverse transcriptase) to the mixture and incubation at 25 °C for 10 min, 42 °C for 40 min and then 70 °C for 15 min. We next created dUTP second strand cDNA by adding the following reaction mixture (10 μl of 5 × second-strand buffer, 0.8 μl of 25 mM dNTPs with 80% of the dTTP replaced by dUTP, 20.5 μl of RNase-free water, 0.2 μl of 5 U/μl RNase H and 2.5 μl of E. coli DNA polymerase I) to the first-strand cDNA mixture, and incubated at 16 °C for 2 h. The double-stranded (ds)DNA products were purified using 1.8 × AMPure beads following the manufacturer’s protocol and end-repaired with a mixture of T4 DNA polymerase, Klenow DNA polymerase and T4 polynucleotide kinase. The end-repaired DNA was then treated with Klenow exo- to add an adenine to the 3’ ends. For paired-end library construction, adaptors were ligated to dsDNA using quick T4 DNA ligase and incubated at 20 °C for 15 min. Subsequently, the ligated DNA product was purified twice with AMPure beads. The adaptor-ligated cDNA was mixed with 1 μl of 10 μM Illumina PE1.0, 1 μl of 10 μM index primer, 1 μl of 50 mM MgSO4, 2.5 μl of 10 × Pfx buffer, 0.4 μl of 25 mM dNTPs, 0.4 μl of 2.5 U/μl Platinum Pfx DNA polymerase (Thermo Fisher, 11708-013) and 3 μl of USER enzyme (NEB, M5505S). The mixture was incubated at 37 °C for 15 min to allow the USER enzyme to digest the dUTP strand, then at 94 °C for 2 min. PCR was performed with the following programme: 11 to 13 cycles of 94 °C for 15 s, 62 °C for 30 s and 72 °C for 30 s. The 200 to 450 bp of PCR products were purified from agarose gel by Qiaqucik mini Elute Kit. The DNA concentration was measured by Qubit and sequenced by Illumina paired-end sequencing.
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Library strategy |
OTHER |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
HiSeq X Ten |
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Data processing |
Library strategy: RIC-seq
Illumina bcl2fastq conversion software (v.2.16) was used for base calling.
After the sequencing of RIC libraries, adapters and PCR duplicates were trimmed off using the Trimmomatic program (v.0.36) (Bolger et al., 2014) and homemade scripts, respectively. The poly(N) tails at the 3’ end were further clipped using the Cutadapt program (v.1.15) (Martin, 2011).
After filtering, the paired reads were first aligned to 45S pre-rRNA, and the unmapped reads were further aligned to the human reference genome (assembly version: hg19) using the STAR software (v.020201) (Dobin et al., 2013). The parameters used were as follows: STAR --runMode alignReads --genomeDir index --readFilesIn read.fq --outFileNamePrefix outprefix --outFilterMultimapNmax 100 --outSAMattributes All --alignIntronMin 1 --scoreGapNoncan -4 --scoreGapATAC -4 --chimSegmentMin 15 --chimJunctionOverhangMin 15. Three additional parameters were further applied (--alignSJoverhangMin 15 --alignSJDBoverhangMin 10 --alignSJstitchMismatchNmax 5 -1 5 5) to improve the mapping quality.
The low-quality and secondary mapping results were filtered using the SAMtools package (v.0.1.19).
Genome_build: hg19
Supplementary_files_format_and_content: genome-wide RNA-RNA interaction in hic format produced by Juicebox
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Submission date |
Feb 26, 2019 |
Last update date |
May 05, 2020 |
Contact name |
Yuanchao Xue |
E-mail(s) |
ycxue@ibp.ac.cn
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Organization name |
Chinese Academy of Sciences
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Department |
Institute of Biophysics
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Lab |
Key Laboratory of RNA Biology
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Street address |
Datun Road #15
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City |
Beijing |
ZIP/Postal code |
100101 |
Country |
China |
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Platform ID |
GPL20795 |
Series (1) |
GSE127188 |
RIC-seq for global in situ profiling of RNA-RNA spatial interactions |
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Relations |
BioSample |
SAMN11022210 |
SRA |
SRX5431516 |
Supplementary file |
Size |
Download |
File type/resource |
GSM3629917_pcp_minus_HeLa_Total_rep1.hic |
102.9 Mb |
(ftp)(http) |
HIC |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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