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| Status |
Public on May 17, 2024 |
| Title |
DMS seq WT D3 rep2 |
| Sample type |
SRA |
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| Source name |
129S1/CastEiJ x 129S1
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| Organism |
Mus musculus |
| Characteristics |
cell line: 129S1/CastEiJ x 129S1
cell type: Day 3 differentiated mouse embryonic stem cells
genotype: WT
treatment: None
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| Treatment protocol |
Trypsinized ~ 2 x 107 cells were resuspended in the 5 ml culture medium and quickly transferred to the chemical fume hood. 200 ul (4% v/v) of DMS is directly applied to the media using a 1 ml syringe. The mixture was incubated at 37°C for 7 min with mild agitation. DMS modification was quenched by adding 1.3 ml βME after putting the tubes on ice. Cells were recovered by centrifugation at 400 g and 4 °C for 3 min. For complete removal of remaining DMS, cell pellets were repeatedly washed twice with 5ml ice-cold 1x PBS and 25% βME buffer. DMS-treated cells are stored in a – 80 °C deep freezer until the RNA purification.
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| Growth protocol |
Clonal TsixTST/+ mESC (TST) line was cultured as routine procedure. mESC were grown on inactivated feeders in high glucose D-MEM supplemented with 15% Hyclone FBS, 25 mM HEPES pH 7.2-7.5, 1x MEM non-essential amino acids, 1x Pen/Strep, 0.1 mM βME, and 500 U/mL ESGRO recombinant mouse Leukemia Inhibitory Factor (LIF) protein at 37°C with 5% CO2. EB differentiation was performed with growing feeder-depleted cells in the medium without LIF. Cells were kept in suspension until 3 days of differentiation and transferred to gelatinized plates on day 3 for further adherent growth.
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| Extracted molecule |
other |
| Extraction protocol |
Frozen cells pellets are placed on dry ice, resuspended in nuclei isolation buffer (15 mM Tris-HCl pH 7.5, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 250 mM Sucrose, 0.3 % Igepal CA-630, 0.1% βME and 10 mM ribonucleoside vanadyl complex). The solution was repeatedly pipetted until the frozen pellet is completely dissolved. Nuclei were collected at 600 g and 4 °C for 5 min and washed twice with the nuclei isolation buffer without Igepal CA-630. To remove soluble nuclear fraction, nuclei pellet was resuspended in 500 ul NUN1 buffer (20 mM Tri-HCl, pH 8.0, 75mM NaCl, 0.5 mM EDTA, 50% Glycerol and proteinase inhibitor 1xComplete followed by 4.8 ml NUN2 buffer (20 mM HEPES-KOH pH 7.6, 7.5 mM MgCl2, 0.2 mM EDTA, 300 mM NaCl, 1 M Urea, 1% Igepal CA-630). The solution was incubated on ice for 15 min with brief vortexing every 5 mins. An insoluble chromatin pellet was obtained by centrifugation at 10000 g at 4 °C for 15 min centrifugation. 300 ml Trizol was added to the chromatin pellets, and under freeze-thaw cycles three times. Then the pellets were ground with eppendorf tube pestles until completely resuspended in trizol. Additional 700 ml Trizol was applied on the top of the ground mixture and the remaining RNA isolation procedure was performed as following standard Trizol protocol. Poly A+ RNA was collected from 100 - 250 ug chromatin-bound RNA using Oligotex Direct mRNA kit.
1 - 5 ug of poly A+ RNA was chemically fragmented at 95 °C for 8 min in RNA fragmentation buffer (100 mM Tris-HCl pH 8 and 2 mM MgCl2). The fragmented RNA was ethanol precipitated, resuspended in urea gel loading buffer (1x TBE, 3.5M Urea, 0.005% Bromophenol blue, and 0.025% Xylene), and subjected to denaturing urea 8% acrylamide gel fraction. 60- 80 nt RNA fragment was excised and recovered from the gel. RNA samples are dephosphorylated with polynucleotide kinase (PNK) (NEB M0201S) in 10 ml solution comprising 1 ml 10x T4 RNA ligase buffer (NEB, B0216), 1.5 ml T4 PNK and 0.5 ml SUPERase•In RNase Inhibitor (Invitrogen, AM2696) at 37 °C for 5 hours. The samples are ligated to the 3’ adaptor by directly adding 6 ml 50 % PEG8000, 1 ml 10x T4 RNA ligase buffer, 2 ml 20 mM adenylated 3 DNA adaptor, and 1 ml T4 RNA Ligase 2 truncated K227Q (NEB, M0373) to the dephosphorylated mixture above and incubating 16 °C for 16 hours. Samples are ethanol precipitated and gel fractionated again, adaptor-ligated products appearing at 85 -105 nt position were gel-purified and resuspended in 13 ul water. For reverse transcription, 13 ml adaptor-ligated RNA was mixed with 4 ml 5x RT buffer (100 mM Tris-HCl pH 7.5, 750 mM KCl (or LiCl), 15 mM MgCl2), 1 ml 10 mM dNTP mix, 2 ml 10 mM phosphorylated iSp18 PE Reverse oligo and 0.1 ul SUPERase•In RNase Inhibitor. The mixture was incubated in PCR machine at 80 °C for 2 min and then 42 °C for 5min, 1 ml 0.1M DTT and 1 ml SuperScript III reverse transcriptase (Invitrogen, 18080085) was added to the mixture while PCR tubes are in the thermocycling block. After incubation at 42 °C for 10 min for cDNA synthesis, samples are moved on ice and then 2 ml 1 M NaOH was added to the reaction mixture. To hydrolyze RNA, samples were treated at 98°C for 15 minutes, and 2 ml 1 M HCl was applied to neutralize the solution. Resulting cDNAs were ethanol precipitated and urea gel fractionated, smearing band appearing at the 78 -105 nt length was purified from the gel. cDNA samples are circularized using 50 U CircLigase (Epicentre, CL4111K) at 60°C for 12 hours. Circularized cDNA was amplified using RT stop amp primer (Supplementary Table 7) and KAPA HiFi PCR master mix (Roche, KK2601) for 8–11 cycles of PCR. Second size selection was performed with native TBE- 8% acrylamide gel fractionation, PCR bands at 78- 105 bp are purified from the gel. 4 - 5 cycles of additional PCR reactions were run with NEBnext multiplex oligos (NEB, E7335) to introduce Illumina library sequences to the RT-stop profile library. ZR small-RNA PAGE Recovery Kit (Zymo Research, R1070) and SYBR Gold (Invitrogen, S11494) staining was used for gel purification during the preparation process.
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| Library strategy |
OTHER |
| Library source |
other |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
3’ end adaptor sequences (-a AGATCGGAAGAGC) are trimmed from raw data files using cutadapt followed by duplicate removal by prinseq. 6 nt molecular barcodes were further clipped away with -g ^NNNNN argument of cutadapt. RT-stop reads out of the range of 18- 45 nt length were excluded from the final fastq file using prinseq -max_len 45 -min_len 18 command.
For RT-stop profile, processed sequencing reads are aligned with STAR aligner against GRCm38.p6 genome assembly. 1 mismatch was allowed for the read mapping. The subsequent sam files are converted to sorted bam files using samtools, RT-stops at the rG4 regions were counted using the rG4seeker pipeline. In vitro rG4 identification was computed from RT-stop counting of two biological replicates. Unknown and G>=40% classes are excluded from the assay due to their absence of rG4 motifs. In vivo rG4 DMS sensitivity was addressed by analyzing K+ dependent RT-stop frequency changes of DMS treated samples.
Assembly: mm10
Supplementary files format and content: "rG4 RT-stop count" Comma-separated files (.csv) indicate rG4 location information (Chr, start, end, class, genome position, splicing site, motif sequence, gene name, and annotation) and RT sequencing read coverage and stops in the K+ and Li+ samples.
Library strategy: DMS-seq
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| Submission date |
Dec 05, 2023 |
| Last update date |
May 17, 2024 |
| Contact name |
yongwoo lee |
| E-mail(s) |
ylee@molbio.mgh.harvard.edu
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| Phone |
6177552856
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| Organization name |
MASSACHUSETTS GENERAL HOSPITAL
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| Department |
Mol bio
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| Lab |
Jeannie Lee Lab
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| Street address |
185 Cambridge St
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02114 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE219079 |
Epigenetic regulation by dynamic RNA G-quadruplex folding and unfolding (DMS-Seq) |
| GSE219083 |
G-quadruplex folding in Xist RNA antagonizes PRC2 activity for step-wise regulation of X-chromosome inactivation |
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| Relations |
| BioSample |
SAMN38677058 |
| SRA |
SRX22782397 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7946723_rG4_RT-stop_count_WT_D3_BR2_rG4.csv.gz |
286.3 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
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