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| Sample GSM4609329 | Query DataSets for GSM4609329 |
| | Status | Public on Jun 11, 2020 | | Title | AB863M rep4 | | Sample type | SRA | | Source name | fixed human breast cancer PDTX tissue | | Organisms | Drosophila melanogaster; Homo sapiens; Mus musculus | | Characteristics | cell type: breast cancer tissuestrain: NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mouse strainchip antibody: BG4 single-chain antibodyspike-in reference organsms: Drosophila melanogasterspike-in cell line: Drosophila S2 cells | | Extracted molecule | genomic DNA | | Extraction protocol | PDTX chromatin prep and G4-ChIP-seq: Briefly, D. melanogaster S2 cells were cultured in Schneider's Drosophila Medium (Thermo Fisher Scientific, cat no. R69007) containing 10% fetal bovine serum (FBS) Medium (Thermo Fisher Scientific, cat no. 10500064). To prepare spike-in Drosophila chromatin, 100 million cells were i) harvested by centrifugation, ii) fixed for 10 min in a solution of media containing 10 % FBS, 1% Formaldehyde (Thermo Fisher Scientific, cat no. 28908) and iii) quenched for 5 min by addition of 125 mM glycine (Fisher Scientific, cat no. 11545005). The cell pellet was washed with 10 mL PBS, pelleted by centrifugation and subsequently stored on ice for the lysis procedure. The 2-step chromatin lysis procedure was performed according to the Chromatrap procedure ("Spin column ChIP kit for qPCR v6.4"). 500 µL intact chromatin was sonicated into 100-500 bp fragments using a Bioruptor Plus (Diagenode cat. no. B02010003 with cooling) at 4C. Sonicated chromatin was diluted with 1.5 mL lysis buffer (Chromatrap cat no. 100005) before snap-freezing as 25 µL aliquots. PDTX chromatin was prepared essentially as described in Schmidt et al. (Methods 2009)30. Briefly, a snap-frozen PDTX biopsy, ~1 cm3, was transferred into a 50 mL falcon tube, on dry-ice, and crushed into smaller chunks on dry-ice using a scalpel followed by fixation for 20 min in 30 ml solution A, containing 1% formaldehyde, and then quenched for 5 min by adding 125 mM glycine. The supernatant of the pelleted tissue was discarded, and the pellet washed twice with 10 mL ice-cold PBS before resuspending in 1 mL PBS and transferred to a 1 mL glass Douncer (Fisher scientific, cat no. 11591295). 10 strokes were employed for each douncing step with a loose and then tight pestle, and the remaining tissue slurry was transferred to a 15 mL tube, centrifuged for 5 min at 2500xg and subjected to lysis according to Schmidt et al30. Briefly, after the 10 mL LB2 treatment and nuclei pelleting step, the pellet was resuspended in 500 µL LB3 and LB3-chromatin solution split into two Bioruptor TBX (Diagenode, cat no. C30010010-300) sonication tubes. The samples were sonicated until the desired fragment length (100-500 bp) was achieved. Finally, 50 µL of a 10% Triton X-100 LB3 solution was mixed with the sonicated solution and aliquoted into 50 µL aliquots before snap-freezing in liquid nitrogen. 5 μL of PDTX chromatin was quantified by Qubit using the "broad range kit" (Thermo Fisher Scientific, cat. no. Q32853). In each qG4-ChIP-seq reaction, 225ng of PDTX chromatin, 102ng of spike-in drosophila chromatin and 2% RNaseA (Invitrogen, cat. no. AM2271) in blocking buffer (25mM HEPES, pH 7.5, 10.5mM NaCl, 110mM KCl, 1mM MgCl2 and 1% BSA (Merck, cat. no. A7030) in Milli-Q water were mixed and incubated at 37C for 30min at 800rpm. All PDTX chromatin batches containing a different concentration than 30 ng/μL were balanced to the same level, either by dilution with LB3 containing 1% Triton X-100 or by up-scaling the ChIP reaction. For PDTX chromatin with a concentration of 30 ng/μL, 7.5 μL of the PDTX chromatin was added to a solution containing 270 μL blocking buffer including 2% RNase A and 7.5 μL spike-in drosophila chromatin. After RNaseA treatment, 15 μL of 2M BG4, prepared as described previously31, was added to each qG4-ChIP-seq reaction and the reaction mixture shaken at 1,400 rpm at 16C for 1 hour. Meanwhile, 65 μL of anti-FLAG magnetic beads (Sigma-Aldrich, cat. no. M8823) were washed three times with 650 μL of blocking buffer and resuspended in 1,300 μL blocking buffer. The pre-washed beads were incubated at 16C at 1,400rpm and 300 μL of pre-washed beads added to the reaction mixture after BG4 incubation. The reaction mixture with beads was incubated at 16C for 1 hour at 1,400rpm. Then, the beads were washed four times in 400 μL cold wash buffer (100mM KCl, 0.1% Tween 20 and 10mM Tris, pH 7.4 in Milli-Q water) in the cold room and twice at 37C for 15min at 1,400rpm, followed by one cold wash on magnetic stand. The enriched chromatin on beads was resuspended in 75 μL TE buffer and 1 μL Proteinase K (Invitrogen, cat. no. AM2546) added. 6 μL Proteinase K was added to input sample which refers to a qG4-ChIP-seq reaction mixture without BG4 and beads. The reaction mixture was incubated at 65C for 3 hours at 1,400 rpm and purified using QIAGEN MinElute Kit (QIAGEN, cat. no. 28206).Purified DNAs were subjected to Nextera DNA library preparation.For 40 μL library preparation reaction, 3-5ng of the ChIP or input DNA (Qubit high sensitivity kit, Thermo Fisher Scientific, cat. no. Q32854), 20 μL 2X tagmentation buffer (Illumina, cat. no. 15027866), 1.25 μL Tn5 enzyme (Illumina, cat no. 18027865) and nuclease-free water was incubated at 37°C for 20min at 800rpm. The reaction mixture was purified using QIAGEN MinElute Kit (QIAGEN, cat. no. 28206) according to the manufacturer’s instruction and eluted in 20 μL EB buffer. To amplify the library, 20 μL of the DNA was then mixed with 25 μL NEB Next High Fidelity 2X PCR Master Mix (New England Biolabs, cat. no. N0541S), 2.5 μL Nextera index kit i5 primer (Illumina, cat. no. 15055290) and 2.5 μL Nextera index kit i7 primer (Illumina, cat. no. 15055290). The PCR program was as follows: 72°C for 5 minutes, 98°C for 30 seconds, followed by 8 cycles of 98°C for 10 seconds, 63°C for 30 seconds and 72°C for 1 minute. Libraries were quantified using a Bioanalyzer (Agilent) to estimate the average library size and concentration determined via Qubit HS. The library concentration was corrected for the library size using the following relationship:1 ng/µL = 3nM = 500 bp. Samples were subjected to single-end sequencing with a read length of 75bp on an Illumina NextSeq instrument.” | | Library strategy | ChIP-Seq | | Library source | genomic | | Library selection | ChIP | | Instrument model | Illumina NextSeq 500 | | Data processing | Fastq files were trimmed from adapters using cutadapt (options: -q 20 -O 3 http://dx.doi.org/10.14806/ej.17.1.200, ver: 1.16) and aligned32 to a combined genome consisting of hg19 (H. Sapiens), dm6 (D. melanogaster) and mm10 (M.musculus) with bwa-mem (ver. 0.7.17-r1188). Bam files were generated from the alignment with samtools view (options: -Sb -F2308 -q 10, ver: 1.9) and subsequently split by organisms to obtain 3 bam files for each sample.Duplicated reads were marked and removed using picard MarkDuplicates (ver: 2.20.3).For all organisms, the total sequencing coverage (total recovery) was quantified as the total number of unique reads aligning to the respective genomeStandard peak calling was performed for each sample using MACS2 (ver. 2.1.2) with default options on hg19 and dm6 bam files respectively.For each human PDTX model, peak regions were considered positive if confirmed in 2 out of 4 technical replicates (multi2) with bedtools v2.27.1 multiinter. All human confirmed G4-ChIP-seq peak files (multi2) of the 22 models were merged (bedtools merge) and regions more than 99 bp long retained to generate a single G4 DNA consensus of 26,103 G4 regions. Finally, the coverage of the samples was quantified over the consensus human set (bedtools coverage).Reference normalization factor estimation and human ChIP signal normalization. For each PDTX biopsy, four technical qG4-ChIP experiments were performed and sequenced alongside one input chromatin (control). In each experiment, a similar amount of reference (D. melanogaster) chromatin from the same batch was added. To estimate PDTX normalization factors, reference coverage was determined at a pre-defined consensus consisting of 1,367 intervals (see Supplementary Data 2). The reference consensus set was defined from, and covers, G4-enriched regions observed in more than 110 pull-down experiments. The normalization factor of each ChIP sample has been defined as the ratio between the maximum observed coverage (across all ChIP samples) and the individual sample coverage. Note that only ChIP experiments were used for this step (i.e. inputs are excluded and forced to 1). . The normalization factors were then exported and used as input for a customized R script performing the normalization of the human signal. For each G4-ChIP-seq experiment, human signal (i.e. read coverage within human G4 consensus) was quantified by performing input subtraction and normalization with their respective reference reads and human library sizes. To assess if the normalization step has globally improved the experimental reproducibility, a quantitative parameter, the Improvement Factor IF, was devised that measures both the increase (i.e. improvement) in data similarity between experiments corresponding to the same technical and biological samples and the increase dissimilarity between different samples. Specifically, the improvement factor of each biological sample has been estimated as: IF = sum_N(D_ratio_inter) - sum_N(D_ratio_intra). Where: o D_ratio: euclidean_distance_after_normalization/euclidean_distance_before_normalization; o N: total N. ChIP samples o euclidean_distance_after_normalization: Euclidean similarity matrix computed on input subtracted, library size adjusted, drosophila normalized data and rescaled to its maximum value; o euclidean_distance_before_normalization: Euclidean similarity matrix computed on input subtracted, library size adjusted data and rescaled to its maximum value; o D_ratio_inter: similarity values among samples belonging to the same technical or biological group; o D_ratio_intra: similarity values among samples not belonging to the same technical or biological group; 135 individual samples (ChIP + Input) were processed from 22 different PDTX models. Some PDTX models have more than one biological sample.Genome_build: hg19, dm6, mm10Supplementary_files_format_and_content: bigwig files *nodup.w10.rpm.bw were generated from the duplicates removed bam files binning the regions in 10bp windows and normalizing the coverage in those regions by the total number of unique reads. The bigwig files *nodup.dm6.sorted.bw were generated starting from **nodup.w10.rpm.bw and following those steps: 1. bw converted to begraph; 2.using bedtools unionbedg perform input subtraction (pull-down minus input RPM) and multiply by drosophila normlization factors. | | Submission date | Jun 10, 2020 | | Last update date | Jun 11, 2020 | | Contact name | Angela Simeone | | E-mail(s) | [email protected] | | Organization name | Genomic Services QIAGEN | | Street address | 200 Hathersage Rd | | City | Manchester | | ZIP/Postal code | M13 0BH | | Country | United Kingdom | | Platform ID | GPL28658 | | Series (1) | | GSE152216 | Landscape of G-quadruplex DNA structural regions in breast cancer |
| | Relations | | BioSample | SAMN15202196 | | SRA | SRX8521366 |
| Supplementary file | Size | Download | File type/resource | | GSM4609329_863_317_R4.all.hg19.merged.nodup.dm6.sorted.bw | 331.2 Mb | (ftp)(http) | BW | | GSM4609329_863_317_R4.all.hg19.merged.nodup.w10.rpm.bw | 164.1 Mb | (ftp)(http) | BW | SRA Run Selector | | Raw data are available in SRA | | Processed data provided as supplementary file |
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