DNA samples were first tested for concentration and integrity, and only samples that passed this test were used for library preparation, and then genomic DNA wsa randomly fragmented by sonication using the Covaris method. Subsequently, genomic DNA fragments with an average length of 200 to 400 base pairs were isolated using the agencourt AMPure XP-Medium kit. The selected genomic DNA fragments were end-repaired and 3′ adenylated, and then ligated to the adenylated fragments. The ligated DNA product was amplified by PCR and purified using the agencourt AMPure XP-Medium kit. After thermal denaturation (95 °C, 1 min), the purified PCR product was cyclized using the ligated oligonucleotide sequence to yield single-stranded circular DNA (ssCir DNA). This ssCir DNA constituted the final library and was rigorously characterized by quality control procedures. Libraries meeting quality standards were sequenced using the MGISEQ-2000 sequencer. ssCir DNA molecules were circularly replicated to form a DNA nanoball (DNB) containing more than 300 replicate copies. These DNPs were then loaded onto patterned nano-arrays using advanced high-density DNA nano-chip technology. Ultimately, 150 base pair reads were generated from the other end of the ssCir DNA using the combinatorial probe anchor synthesis (cPAS) method.
Ampure xp medium kit
Manufactured by Beckman Coulter
The AMPure XP-Medium kit is a reagent kit used for the purification of nucleic acids, such as DNA and RNA, from various sample types. The kit utilizes carboxylate-coated magnetic beads to selectively bind and capture nucleic acids, allowing for efficient removal of contaminants and unwanted molecules. This process facilitates the isolation and concentration of the target nucleic acids for downstream applications.
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Lab products found in correlation
2100 bioanalyzer, by Agilent Technologies (4 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Ultrasonic high performance sample processing system, by Covaris (1 mentions)
Adaptors, by Illumina (1 mentions)
Qiaamp dna microbiome kit, by Qiagen (1 mentions)
Hiseq platform, by Illumina (1 mentions)
Axyprep mag pcr clean up kit, by Covaris (1 mentions)
Agilent 2100 bioanalyzer instrument, by Agilent Technologies (1 mentions)
Eland, by Illumina (1 mentions)
Blood genomic dna extraction kit, by Tiangen Biotech (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Anti h3k27me3, by Cell Signaling Technology (1 mentions)
Bgiseq 500 platform, by BGI Group (1 mentions)
Anti h3k27ac, by Abcam (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Qubit rna assay kit, by Thermo Fisher Scientific (1 mentions)
Nanophotometer, by Implen (1 mentions)
Bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
Rna nano 6000 assay kit, by Agilent Technologies (1 mentions)
Fast dna spin extraction kit, by MP Biomedicals (1 mentions)
25 protocols using ampure xp medium kit
1
Robust Single-Cell Genomic Library Construction
2
Whole-Genome DNA Library Preparation
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After extraction of the genomic DNA, we generate random fragments of the genomic DNA by using ultrasonicator (Covaris). Then, the genomic DNA fragments with an average size of 200–400 bp were obtained with Agencourt AMPure XP-Medium kit. Selected fragments of genomic DNA were end repaired and 3′ adenylated, followed by adaptor ligation to the ends of these 3′ adenylated fragments. The ligation DNA products were amplified by PCR and purified by the Agencourt AMPure XP-Medium kit. The purified PCR products were denatured to single strand DNA by heating, and circularised by the splint oligo sequence. The single strand circle DNA (ssCir DNA) were regarded as the final library and qualified by QC. We sequenced the final qualified libraries by using MGISEQ-2000 sequencer. An ssCir DNA molecule formed a DNA nanoball (DNB), which contains more than 300 copies by rolling-cycle replication. We loaded the DNBs into the patterned nanoarray by using high density DNA nanochip technology. In the end, we obtained pair-end 150 bp reads by using combinatorial Probe-Anchor Synthesis (cPAS).
3
Whole-Exome Sequencing Protocol
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Whole-exome sequencing was performed by the Beijing Genomics Institute. Genomic DNA (1 μg) was first randomly fragmented by Covaris. Fragmented DNA was selected by an Agencourt AMPure XP-Medium Kit to an average size of 200 to 400 bp. The selected fragments were end-repaired, 3′-adenylated, adapter-ligated, and PCR-amplified and the products were recovered using the AxyPrep Mag PCR Clean-Up Kit. The PCR products were hybridized with Agilent Hybridization and Wash Kits. After that, the AxyPrep Mag PCR Clean-Up Kit was used to recover the products as before. The double-stranded PCR products were heat-denatured and circularized by the splint oligo sequence, forming the final library of single-stranded circular DNA. The library was amplified to make DNA nanoballs (DNBs). The DNBs were loaded into the patterned nanoarray and paired-end 100 base reads were generated by sequencing with combinatorial probe–anchor synthesis on the DNBseq platform.
4
Whole Blood Genomic DNA Sequencing
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Genomic DNA was extracted from whole blood obtained from the patients and any participating family members. DNA samples were randomly disrupted by the ultrasonic high-performance sample processing system (Covaris). Segments were selected using the Agencourt AMPure XP-Medium kit, and library preparation was performed. Then the DNA of the target gene exon and adjacent splice regions was captured and enriched using a BGI V4 chip. Sequencing was performed on the Mgiseq-2000 sequencing platform. The average effective sequencing depth of the target region was ≥100X, and >95% of target bases, and had coverage >20X. The sequences were compared with the UCSC hg19 human reference genome by BWA. SNP detection and InDel analysis were performed, and then the mutation was selected by filtering through database annotation. The classification of variant pathogenicity is based on the American College of Medical Genetics (ACMG) guidelines.
5
Metagenomic Shotgun Sequencing Protocol
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All samples were prepared for the metagenomic shotgun sequencing according to previous protocols [6 (link)]. Briefly, total genomic DNA was extracted using QIAamp DNA Microbiome Kit (Qiagen, USA). After DNA extraction, 1 µg genomic DNA was randomly fragmented by Covaris, followed by purification by AxyPrep Mag PCR clean-up kit. The fragmented DNA was selected by Agencourt AMPure XP Medium kit to an average size of 200–400 bp. The fragments were end-repaired by End Repair Mix and purified afterward. The repaired DNAs were combined with A-Tailing Mix. Then the Illumina adaptors were ligated to the Adenylate 3’Ends DNA and followed by purification. The products were selected based on the insert size. Several rounds of PCR amplification with PCR Primer Cocktail and PCR Master Mix were performed to enrich the Adapter-ligated DNA fragments. After purification, the library was qualified by the Agilent 2100 bioanalyzer (Agilent, USA) and ABI StepOnePlus Real-time PCR System. Finally, the qualified libraries were sequenced on the Illumina Hiseq platform (BGI-Shenzhen, China).
6
Whole Genome Sequencing Workflow
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Genomic DNA was isolated from venous blood according to a previously described standard protocol [56 (link)] with slight modifications [57 (link)] and prepared according to the requirements of the WGS service provider (BGI Genomics, Hong Kong). All samples passed quality control measures stipulated by BGI Genomics. In summary, for each sample the isolated DNA was fragmented, and the fragments selected by Agencourt AMPure XP-Medium kit to an average size of 200-400bp. Fragments were end repaired and 3’ adenylated and adaptor sequences ligated to the 3’ adenylated fragments. Fragments were then amplified by PCR and underwent a purification step followed by heat denaturation and circularized. Single stranded circular (ssCir) DNA formatted to a library were qualified by QC measures and sequenced using the BGISEQ-500 at 30X coverage. ssCir DNA molecule nanoballs were loaded into a patterned nanoarray using high density DNA nanochip technology, and pair end 100bp reads obtained by combinatorial Probe-Anchor Synthesis (cPAS).
7
DNA Fragmentation and Library Preparation
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Genomic DNA (1 μg) was randomly fragmented by Covaris. The fragmented DNA was selected by an Agencourt AMPure XP-Medium Kit to an average size of 200–400 bp. The selected fragments were subjected to end repair, 3′ adenylation, adaptor ligation, and polymerase chain reaction (PCR) amplification, with the products, and then being recovered using an AxyPrep Mag PCR Clean-up Kit. The double-stranded PCR products were heat denatured and circularized by the splint oligo sequence. Single-stranded circular DNA (ssCir DNA) was formatted as the final library and quality controlled. The quality-controlled libraries were sequenced on the MGISEQ2000 platform.
8
Transcriptome Profiling of Rat Microglia
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Total RNA was extracted from the isolated microglia from the brain using TRIzol reagent. Subsequently, the total RNA was identified and the full-length cDNA was amplified by PCR. The average molecule length was determined using the Agilent 2100 bioanalyzer instrument (Agilent High Sensitivity DNA Reagents). Library was constructed following Tagmentation-based library construction protocol. PCR products were purified and selected with the Agencourt AMPure XP-Medium kit. DNA was quantified by Agilent Technologies 2100 bioanalyzer. Library was qualified by the Agilent Technologies 2100 bioanalyzer. The library was amplified to make DNA nanoball (DNB) which had more than 300 copies of one molecular. The DNBs were loaded into the patterned nanoarray and single end 50 bases reads were generated in the way of sequenced by combinatorial Probe-Anchor Synthesis (cPAS). The raw sequencing data were aligned to the rat genome. Differentially expressed genes (DEGs) were identified by PossionDis analysis with FDR < 0.001.
9
ChIP and qChIP Assays in MCF-7 Cells
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ChIP and qChIP assays were performed using MCF-7 cells as described previously39 (link). Briefly, 1 × 107 cells were crosslinked with 1% formaldehyde, sonicated, precleared, and incubated with 2–3 μg of primary antibody against normal rabbit IgG (control), GATA3, or UTX. The complex was washed with low-salt and high-salt buffers and then DNA was extracted for qChIP and ChIP-seq assays. The primers used for qChIP are listed in Supplementary File 3 . For ChIP-seq, quantified 10 ng of DNA was resolved using an Agilent Technologies 2100 Bioanalyzer and 50–250-bp fractions were extracted, and the fractions were then subject to end-repair and 3ʹ-adenylation. Adapter-ligated libraries were amplified, purified, and selected using an Agencourt AMPure XP-Medium kit, and the final library was composed of single-stranded circular DNAs. In-depth whole-genome DNA sequencing was performed by CapitalBio Corporation (Beijing, China). Sequencing data acquired from the Illumina analysis pipeline were compared with the unmasked human reference genome hg19 (UCSC GRCh37) by using ELAND (Illumina, San Diego, CA, USA). The peaks were called using Model-based Analysis of ChIP-Seq (MACS) after filtering through the input. ChIPseeker was used to analyze genomic distribution of GATA3 or UTX binding sites.
10
Whole Genome Sequencing of Trios
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We sequenced the patient and her parents following the MGI-2000 protocol outsourced to BGI. Genomic DNA was isolated from peripheral blood using a blood genomic DNA extraction kit (Tiangen Biotech, Beijing, China) in accordance with the manufacturer’s protocol. One microgram of genomic DNA was randomly fragmented by Covaris, and the fragmented DNA was selected by an Agencourt AMPure XP-Medium kit to an average size of 200–400 bp, followed by adapter ligation and PCR amplification. The products were recovered by the AxyPrep Mag PCR clean up Kit. The double-stranded PCR products were heat-denatured and circularized by the splint oligo sequence. The single-strand circle DNA (ssCir DNA) was formatted as the final library and qualified by QC. WGS was performed on the MGI-2000 platform with an average depth of 30x, meaning that the entire genome was sequenced an average of 30 times.
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