5′ biotinylated probes (see Table S2 ) were designed using the online tool by the Hughes lab (CapSequm, http://apps.molbiol.ox.ac.uk/CaptureC/cgi-bin/CapSequm.cgi ) to be 70-120bp long and two probes for each promoter of interest. The probes were pooled at 2.9nM each. Samples were captured twice and hybridizations were carried out for 72h and for 24h for the first and the second captures, respectively. To even out capture differences between tubes, libraries were pooled prior to hybridization. For Control, SCC1DEG, RING1BDEG and SCC1DEG RING1BDEG, 1.5μg of each replicate was individually hybridized and then pooled for the second round of hybridization. CTCF ± AUX were multiplexed prior to the first capture at 2 μg each. Hybridization was carried out using Nimblegen SeqCap (Roche, Nimblegen SeqCap EZ HE-oligo kit A, Nimblegen SeqCap EZ HE-oligo kit B, Nimblegen SeqCap EZ Accessory kit v2, Nimblegen SeqCap EZ Hybridization and wash kit) following manufacturer’s instructions for 72 h followed by a 24 h hybridization (double Capture). The captured library molarity was quantified by qPCR using SensiMix SYBR (Bioline, UK) and KAPA Illumina DNA standards (Roche) and sequenced on Illumina NextSeq 500 platform for three biological replicates.
Nimblegen seqcap ez hybridization and wash kit
Manufactured by Roche
The NimbleGen SeqCap EZ Hybridization and Wash Kit is a laboratory product designed to facilitate the hybridization and washing process in next-generation sequencing applications. The kit provides the necessary reagents and protocols to ensure efficient target enrichment and library preparation.
Automatically generated - may contain errors
Lab products found in correlation
Dynabeads m 270, by Thermo Fisher Scientific (6 mentions)
Kapa hifi hotstart readymix, by Roche (5 mentions)
Human cot 1 dna, by Thermo Fisher Scientific (4 mentions)
Ampure xp beads, by Roche (4 mentions)
Xgen universal blocking oligos, by Integrated DNA Technologies (4 mentions)
Sensimix sybr, by Meridian Bioscience (3 mentions)
Nimblegen seqcap ez he oligo kit a, by Roche (3 mentions)
Nimblegen seqcap ez he oligo kit b, by Roche (3 mentions)
Nimblegen seqcap ez accessory kit v2, by Roche (3 mentions)
Nextseq 500 platform, by Illumina (3 mentions)
Kapa library quantification kit, by Roche (3 mentions)
Hiseq 4000 ngs platforms, by Illumina (3 mentions)
2100 bioanalyzer, by Agilent Technologies (3 mentions)
Dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Qubit 3, by Thermo Fisher Scientific (2 mentions)
Dneasy blood tissue kit, by Qiagen (2 mentions)
Xgen lockdown probes panel, by Integrated DNA Technologies (2 mentions)
Nanodrop one, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Hiseq ngs platform, by Illumina (1 mentions)
11 protocols using nimblegen seqcap ez hybridization and wash kit
1
Capture-C Probe Design and Sequencing
2
Targeted DNA Sequencing Library Preparation
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DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany. Purified DNA was qualified by Nanodrop One (Thermo Fisher, Waltham, MA) and quantified by Qubit 3.0 (Life Technologies, Singapore) using the dsDNA HS Assay Kit (Life Technologies, Eugene, OR) according to the manufacturer’s recommendations. Different libraries with unique indices were pooled together in desirable ratios for up to 2 μg of total library input. Human cot-1 DNA (Life Technologies, Carlsbad, CA) and xGen Universal blocking oligos (Integrated DNA Technologies, Coralville, IA) were added as blocking reagents. The capture reaction was performed with the NimbleGen SeqCap EZ Hybridization and Wash Kit (Roche, Madison, WI) and Dynabeads M-270 (Life Technologies, Vilnius, Lithuania) with optimized manufacturers’ protocols. Captured libraries were on-beads amplified with Illumina p5 and p7 primers in KAPA HiFi HotStart ReadyMix (KAPA Biosystems, Cape Town, South Africa). The post-capture amplified library was purified by Agencourt AMPure XP beads and quantified by qPCR using the KAPA Library Quantification kit (KAPA Biosystems). Library fragment size was determined by the Agilent Technologies 2100 Bioanalyzer (Agilent, Santa Clara, CA). The target-enriched library was then sequenced on HiSeq NGS platforms (Illumina, San Diego, CA) according to the manufacturer’s instructions.
3
Capture and Sequencing of Genomic Regions
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The probes described in Supplementary Table S1 were pooled at 2.9 nM each. Samples were captured twice and hybridizations were carried out for 72 h and for 24 h for the first and the second captures, respectively. To even out capture differences between tubes, libraries were pooled prior to hybridization at 1.5 μg each. Hybridization was carried out using Nimblegen SeqCap (Roche, Nimblegen SeqCap EZ HE-oligo kit A, Nimblegen SeqCap EZ HE-oligo kit B, Nimblegen SeqCap EZ Accessory kit v2, Nimblegen SeqCap EZ Hybridization and wash kit) following manufacturer's instructions. The captured library molarity was quantified by quantitative polymerase chain reaction (qPCR) using SensiMix SYBR (Bioline, UK) and KAPA Illumina DNA standards (Roche) and sequenced on Illumina NextSeq 500 platform for 4 (ESC Fbxl19-CxxCf/f), 3 (Med13/13lf/f) or 2 (RA Fbxl19-CxxCf/f) biological replicates.
4
Whole-Exome Capture and Sequencing Protocol
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DNA libraries were subjected to whole-exome capture with xGen Exome Research Panel v1.0 (Integrated DNA Technologies), which spans a 39 Mb target region (19,396 genes) of the human genome and covers 51 Mb of end-to-end tiled space. Human Cot-1 DNA (Life Technologies) and xGen universal blocking oligos (Integrated DNA Technologies) were added as blocking reagents to reduce non-specific hybridization. The capture reaction was conducted with NimbleGen SeqCap EZ Hybridization and Wash Kit (Roche) and Dynabeads M-270 (Life Technologies) according to manufacturer’s recommended protocol. The captured samples were sequenced on an Illumina HiSeq X-TEN platform with a paired-end run of 2×150 bp. The quality of each read was initially verified using the software embedded in the HiSeq X-TEN sequence. A FASTQ file was generated for each tested sample for sequence alignment and converted to a BAM file for further analysis.
5
Targeted Genomic Profiling of FFPE Samples
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According to the manufacturer’s instructions, DNA was extracted by a QIAamp DNA FFPE Tissue Kit (Qiagen, Carlsbad, CA, USA) after twice of de-paraffinized by xylene. Extracted DNA was purified and qualified employing the Nanodrop2000 (Thermo), and then using Qubit3.0 (Life Technology) with a dsDNA HS Assay Kit (Life Technology) to quantify DNA.
Amplified and purified DNA Libraries by PCR and then pooled together 1-2 μg of different libraries for targeted enrichment. Hybridization-based target enrichment was carried out with NimbleGen SeqCap EZ Hybridization and Wash Kit (Roche). Captured libraries by Dynabeads M-270 (Life Technologies) were amplified in KAPA HiFi HotStart ReadyMix (KAPA Biosystems), followed by purification by Agencourt AMPure XP beads. Customized xGen lockdown probes panel (Integrated DNA Technologies) were used to targeted enrich for 425 predefined genes. The enriched libraries were sequenced on Hiseq 4000 NGS platforms (Illumina) to coverage depths of at least 100 × and 300 × after removing PCR duplicates for tumour and normal tissue, respectively.
Amplified and purified DNA Libraries by PCR and then pooled together 1-2 μg of different libraries for targeted enrichment. Hybridization-based target enrichment was carried out with NimbleGen SeqCap EZ Hybridization and Wash Kit (Roche). Captured libraries by Dynabeads M-270 (Life Technologies) were amplified in KAPA HiFi HotStart ReadyMix (KAPA Biosystems), followed by purification by Agencourt AMPure XP beads. Customized xGen lockdown probes panel (Integrated DNA Technologies) were used to targeted enrich for 425 predefined genes. The enriched libraries were sequenced on Hiseq 4000 NGS platforms (Illumina) to coverage depths of at least 100 × and 300 × after removing PCR duplicates for tumour and normal tissue, respectively.
6
Cancer-Relevant Gene Capture and Sequencing Protocol
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Libraries with different indexes were pooled together in optimized ratios to reach up to 2 μg of total DNA. Human Cot-1 DNA (Life Technologies) and xGen universal blocking oligos (Integrated DNA Technologies) were added as blocking reagents to reduce non-specific hybridization. Customized xGen lockdown probes (Integrated DNA Technologies) targeting 5,804 exons of 382 cancer-relevant genes and 37 introns of 16 fusion genes were used for hybridization enrichment (Supplementary Table 1 ). The capture reaction was performed with NimbleGen SeqCap EZ Hybridization and Wash Kit (Roche) and Dynabeads M-270 (Life Technologies) according to manufacturers’ protocols. Post-captured libraries were PCR amplified with Illumina p5 (5′ AAT GAT ACG GCG ACC ACC GA 3′) and p7 primers (5′ CAA GCA GAA GAC GGC ATA CGA GAT 3′) in KAPA HiFi HotStart ReadyMix (KAPA Biosystems), followed by purification with Agencourt AMPure XP beads, and quantified by qPCR method using KAPA Library Quantification kit (KAPA Biosystems). The size distribution of the library was analyzed by Bioanalyzer 2100 (Agilent Technologies). Target-enriched libraries were then sequenced on Illumina MiSeq or HiSeq4000 NGS platforms (Illumina) according to manufacturer's instructions. Targeted capture and sequencing performance of all the samples were summarized in Supplementary Table 3 .
7
Targeted Enrichment of Cancer Genes
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DNA libraries were subjected to polymerase chain reaction (PCR) amplification and purification before targeted enrichment. Libraries from different samples were marked with unique indices during library preparation and up to 2 μg of different libraries were pooled together for targeted enrichment. Human cot-1 DNA (Life Technologies) and xGen Universal Blocking Oligos (Integrated DNA Technologies) were added to block nonspecific binding of library DNA to targeted probes. Customized xGen lockdown probe panels (Integrated DNA Technologies) were used for targeted enrichment of 425 predefined genes. The hybridization reaction was performed using NimbleGen SeqCap EZ Hybridization and Wash Kit (Roche). Dynabeads M-270 (Life Technologies) were used to capture probe-bound fragments. Then, the library was amplified with Illumina p5 and p7 primers in KAPA HiFi HotStart ReadyMix (KAPA Biosystems) and purified using Agencourt AMPure XP beads. Library quantification was achieved using the KAPA Library Quantification kit (KAPA Biosystems). The size distribution was measured using Agilent Technologies 2100 Bioanalyzer (Agilent Technologies). Enriched libraries were sequenced on HiSeq 4000 NGS platforms (Illumina) to coverage depths of at least 100 x and 300 x for normal tissue and tumor, respectively, after removing PCR duplicates.
8
Genomic Profiling of Endometrial Neoplasms
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Genetic testing was performed after pathological diagnosis of EIN and EEC. Genomic DNA was extracted from fresh frozen tissue (somatic) and the matched blood sample (germline) using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. DNA was purified by AxyPrep™ Mag Tissue-Blood genomic DNA Kit (Axygen, America). And its quantification was measured by the Qubit dsDNA HS Assay Kit (Life Technologies, Eugene, OR). All purified DNA samples are judged to be of high quality (concentration > 3.4 ng/ul) for mutation analysis. Fifty nanograms of genomic DNA was fragmented randomly into fragments which size range from 200 to 300 bp. Fragmented DNA was added with barcode and adaptors using polymerase chain reaction (PCR) reagents, the quality of PCR products was checked. The products were used for library construction and follow-up exon capture. Captured fragments were subsequently purified, amplified, ligated, and circularized by NimbleGen SeqCap EZ Hybridization and Wash Kit (Roche NimbleGen, Madison, WI). Finally, high-throughput sequencing of library products was performed on NextSeq CN500 (BerryGenomics, China).
9
Targeted Genomic Capture Sequencing
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5′ biotinylated probes were designed using the online tool by the Hughes lab (CapSequm) to be 70-120bp long and two probes for each promoter of interest. The probes were pooled at 2.9 nM each. Samples were captured twice and hybridizations were carried out for 72 h and for 24 h for the first and the second captures, respectively. To even out capture differences between tubes, libraries were pooled prior to hybridization at 1.5 μg each. Hybridization was carried out using Nimblegen SeqCap (Roche, Nimblegen SeqCap EZ HE-oligo kit A, Nimblegen SeqCap EZ HE-oligo kit B, Nimblegen SeqCap EZ Accessory kit v2, Nimblegen SeqCap EZ Hybridization and wash kit) following manufacturer’s instructions for 72 h followed by a 24 h hybridization (double Capture). The captured library molarity was quantified by qPCR using SensiMix SYBR (Bioline, UK) and KAPA Illumina DNA standards (Roche) and sequenced on Illumina NextSeq 500 platform as 80 bp paired-end reads for three biological replicates.
10
Targeted Enrichment of DNA Libraries
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Amplified and purified DNA Libraries by PCR and then pooled together 1-2 μg of different libraries for targeted enrichment. Hybridization based target enrichment was carried out with NimbleGen SeqCap EZ Hybridization and Wash Kit (Roche). Captured libraries by Dynabeads M-270 (Life Technologies) were amplified in KAPA HiFi HotStart ReadyMix (KAPA Biosystems), followed by purification by Agencourt AMPure XP beads. Customized xGen lockdown probes panel (Integrated DNA Technologies) were used to targeted enrich for 425 predefined genes. The enriched libraries were sequenced on Hiseq 4000 NGS platforms (Illumina) to coverage depths of at least 100x and 300x after removing PCR duplicates for tumour and normal tissue, respectively.
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