We extracted genomic DNA from patient leukemic blasts, from the lymphoid fraction from peripheral blood or bone marrow of remission samples and from bone marrow derived fibroblast cultures using the DNeasy Blood & Tissue Kit (Qiagen). Whole exome sequencing was performed and analyzed as previously described12 . For whole genome sequencing, paired diagnosis, remission, and relapse samples from 15 cases were sequenced on the Illumina HiSeq X Ten System at New York Genome Center (remission at 60x coverage, diagnosis and relapse at 100x coverage), paired diagnosis, remission, and relapse samples from 15 cases were sequenced on the Illumina HiSeq X Ten System at GENEWIZ (remission at 30x coverage, diagnosis and relapse at 60x coverage), and paired diagnosis, remission, and relapse samples from 19 cases were sequenced on the Illumina HiSeq X Ten System at BGI Americas (remission at 30x coverage, diagnosis and relapse at 60x coverage). The analysis produced an average of 90.1 million paired-end reads per sample. After filtering for duplicate reads (i.e. reads with identical start and orientation), sequences were aligned to the reference human genome hg19 assembly using the Burrows-Wheeler Aligner (BWA) tool version 0.7.1532 .
Hiseq x ten system
Manufactured by Illumina
Sourced in United States, China, Japan, United Kingdom
The HiSeq X Ten system is a high-throughput DNA sequencing instrument manufactured by Illumina. The core function of the HiSeq X Ten is to perform large-scale, whole-genome sequencing.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (16 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (15 mentions)
Vahts stranded mrna seq library prep kit, by Vazyme (15 mentions)
Smartspec plus, by Bio-Rad (13 mentions)
Trizol, by Thermo Fisher Scientific (12 mentions)
2100 bioanalyzer, by Agilent Technologies (9 mentions)
Rq1 dnase, by Promega (8 mentions)
Vahts rna adapters, by Vazyme (7 mentions)
Dneasy blood tissue kit, by Qiagen (5 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (5 mentions)
Rneasy mini kit, by Qiagen (5 mentions)
Truseq dna pcr free libraries, by Illumina (5 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (5 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (4 mentions)
Truseq rna sample prep kit, by Illumina (4 mentions)
Truseq rna sample preparation kit, by Illumina (4 mentions)
Rneasy plant mini kit, by Qiagen (4 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Vahts dna clean beads, by Vazyme (4 mentions)
Bioanalyzer 2100 system, by Agilent Technologies (3 mentions)
265 protocols using hiseq x ten system
1
Whole Genome Sequencing of Leukemic Samples
2
Pineapple Genome Sequencing and Transcriptome Analysis
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In this study, plant material of Ananas comosus var. comosus cultivar MD-2 was acquired from Guangxi Academy of Agricultural Sciences (GAAS), China (22.84。N, 108.48。E). Identification of the plant materials was made by the GAAS and original plant was acquired from Del Monte company (www.delmonte.com ). The voucher specimen was deposited at the herbarium of the College of Plant Protection, Fujian Agriculture and Forestry University, China. DNA was extracted from two regions (green tip and white base) of ‘D’ leaf of pineapple (Ananas comosus var. comosus cultivar MD-2) using Qiagen DNeasy Plant Mini Kit, and BS-seq libraries were prepared using the TruSeq Nano DNA LT kit (Illumina), as described previously [32 (link)]. For each tissue type, two libraries corresponding to two biological replicates were sequenced on a HiSeq X Ten system (Illumina) to obtain paired-end 150-bp reads per the manufacturer’s instructions.
Total RNA was extracted from the same tissue used for the BS-seq libraries, and RNA-seq libraries were prepared using the TruSeq Preparation Kit with polyA mRNA selection, per the manufacturer’s instructions (Illumina). Three libraries (only two libraries for 10 am sample) were pooled and sequenced to obtain paired-end 150-bp reads on the Illumina HiSeq X Ten system.
Total RNA was extracted from the same tissue used for the BS-seq libraries, and RNA-seq libraries were prepared using the TruSeq Preparation Kit with polyA mRNA selection, per the manufacturer’s instructions (Illumina). Three libraries (only two libraries for 10 am sample) were pooled and sequenced to obtain paired-end 150-bp reads on the Illumina HiSeq X Ten system.
3
Whole-Exome Sequencing and Variant Analysis
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Whole‐exome sequencing (WES) was performed by parent's request. DNA library was constructed by Agilent SureSelect Human Exon V5 kit (Agilent Technologies) according to the manufacturer's protocols. Sequencing was processed on Illumina HiSeq X Ten System (Illumina, Inc) based on the manufacturer's protocols. The sequencing reads were mapped to the Genome Reference Consortium Human genome build 37 (GRCh37). The Genome Analysis Toolkit (GATK) was used for variant calling. Candidate single nucleotide variants (SNVs) and insertion‐deletions (indels) were saved in VCF files and uploaded to the online variation annotation tool TGex (https://tgex.genecards.cn/#/ ) for further filtering and prioritizing. Common variants were filtered based on the frequencies in the Exome Aggregation Consortium (ExAC) (http://exac.broadinstitute.org ), the Exome Sequencing Project (https://esp.gs.washington.edu ), the 1000G (http://www.1000genomes.org ), genomAD (http://gnomad.broadinstitute.org/ ) and our local database. The variant pathogenicity was assessed according to the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines (Richards et al., 2015 ).
4
Whole-Genome Bisulfite Sequencing of Mouse Samples
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Genomic DNA was purified from cells using DNeasy Blood & Tissue Kit (QIAGEN, 69504). PCR-free WGBS libraries were prepared with the TdT-assisted adenylate connector-mediated ssDNA ligation-mediated Post-Bisulfite Adaptor-Tagging (tPBAT) protocol as described previously (51 (link)). One hundred nanograms of genomic DNA spiked with 1 ng of unmethylated lambda DNA was served for the library preparation. Each library was indexed with different sequences, and sequencing was performed on an Illumina HiSeq X Ten system (Macrogen Japan Corp.) with 2 × 150 paired-end chemistry. The same amounts of libraries prepared from three independent biological replicates were mixed in a tube, and one lane of sequencing was assigned per mix. Note that data of three replicates were merged to increase read depth for the downstream analysis. Sequenced reads were mapped on the reference comprised of mouse mm9 and lambda phage with Bmap as described previously (51 (link)). The alignments were summarized and exported to bedGraphFiles with in-house developed programs described previously (51 (link)). The summary of basic metrics of the DNA methylome data produced in the current study is provided in Supplementary Tables S6 and S7 .
5
Transcriptome Analysis of Porcine Differentiation
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Total RNA was isolated at each time point (D0, D2, D4, and D8) using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). The qualities and quantities of the RNA were measured using Bioanalyzer 2100 (Agilent Technologies, Santa Clare, CA, USA) and 1% agarose gel electrophoresis, which showed that the RNA integrity number (RIN) values of all samples were 10. Ribosomal RNA from each sample was removed using the Ribo-Zero™ GoldKits (Epicentre, Madison, WI, USA). Equal amounts of total RNA of the same stage of differentiation from three Erhualian piglets were pooled into one sample. Then, cDNA libraries were prepared using a NEB Next Ultra Directional RNA LibraryPrep Kit (NEB, Ispawich, MA, USA), according the manufacturer’s instructions and sequenced using the Illumina HiSeq X Ten system (Illumina, San Diego, CA, USA).
6
Genetic Analysis of Hereditary Dermatosis
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To explore the genetic properties of the patients, the capture probe from the Roche NimbleGen Sequence Capture SeqCap EZ Library was used to capture a total of 569 genes associated with hereditary dermatosis. Firstly, DNA was cropped into approximately 300-bp fragments using focused ultrasonicators (Covaris M220, United States) and used to construct the DNA library. Then, streptavidin-coated magnetic beads by NimbleGen (Roche NimbleGen, Inc.) were bound to an avidin-labeled probe after the probe had captured the target exons. Next, a hybridization reaction between the DNA library with various index marks and probes with biotin was performed. After linear PCR amplification, the quality of the library was determined. Sequencing was carried out on an Illumina HiSeq X Ten System (Illumina, San Diego, CA, United States) under sequencing efficiency with an average sequencing depth > 200× and Q30 > 90% according to the manufacturer’s instructions. DNA from all of the probands’ blood samples and tumor tissues from the proband in family 4 were subjected to multigene panel testing.
7
RNA-seq Library Preparation Protocol
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Total RNA extraction, quality inspection, DNase I treatment, and rRNA removal (using the Ribo-Zero kit; Epicentre, Madison, WI, USA) were performed as described previously [25 (link)]. Fragment buffers were added to each sample and the RNA was cut into fragments of 200–500 bp. The first-strand cDNA was synthesized from the RNA fragment using random hammerer primers. Then, using the first strand of cDNA as a template, the second strand of cDNA was synthesized with dUTP instead of dTTP. AMPure XP beads (Beckman Coulter, Brea, CA, USA) were used for fragment size selection and the selected cDNA fragments were amplified via polymerase chain reaction (PCR). Finally, the cDNA library was sequenced using the Illumina HiSeq X Ten system (Illumina, San Diego, CA, USA) from Chengdu Biobaseline Technology Co., LTD. (Chengdu, China).
8
Whole-Exome Sequencing of HCC and Para-Tumor Tissues
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Total DNA from HCC tissues and para-tumor tissues were extracted and subjected to DNA library preparation using QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s instructions. Whole-exome capture was carried out using Agilent SureSelect Human All Exon V6 kits. And sequencing was performed using the Illumina HiSeq X ten system (Annoroad Gene Tech. Co., Ltd). As described previously, qualified WES reads were aligned to hg19 human genome assembly (GRCh37) using BWA, and duplicates of all mapped reads were then marked and discarded using Picard.50 (link)
9
Genomic DNA Extraction and Exome Sequencing Protocol
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Genomic DNA was extracted from the tumor or normal lung samples using a magnetic genomic DNA kit (Tiangen) according to the manufacturer's protocol and from FFPE of tumor and normal samples using an internally modified magnetic extraction protocol. DNA was randomly broken into 150–200 bp using the ultrasonicator (Covaris). The sheared DNA samples were subjected to the end repair step using the Agilent SureSelectXT Low Input Reagent Kit (Agilent), followed by addition of a base A to the 3′ end to form a sticky end. After that, DNA fragments were ligated with specific barcode adapter sequences and any incompletely ligated fragments using the magnetic beads were removed. The samples were then subjected to a PCR amplification using universal primers complementary with adapter sequences to build a DNA sequencing library. The probes from the Agilent SureSelectXT All Human Exome library were utilized to capture target fragments from the samples and the library concentrations were assessed by using Qubit (Thermo Fisher), and the library size was measured using the Agilent TapeStation. The Illumina HiSeq X Ten system (Illumina, Mingma Technologies Co., Ltd) was used to collect data from 150‐bp pair‐end sequencing.
10
Breast Cancer Cell RNA Isolation and Sequencing
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TRIzol reagent (Invitrogen, MD, USA) was used for total RNA extracted from breast cancer cells and their exosomes. Spectrophotometrically 2000 (Thermo Fisher Scientific, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) were used to quantify total RNA. Moreover, all total RNA of breast cell lines and their exosomes was treated with Ribonuclease R (Rnase R) to remove linear RNAs before the construction of RNA-seq libraries. Strand-specific RNA libraries were constructed by using the VAHTS Total RNAseq (H/M/R) Library Prep Kit (Vazyme, China) according to the manufacturer’s protocols for Illumina. Bioanalyzer 4200 (Agilent, USA) was used to analyze purified first-strand cDNA of the libraries through PCR amplification. Finally, the cDNA was sequenced using an Illumina HiSeq X Ten system (Illumina, USA). All data can be found in previous articles published by our team by Dr. Zhong17 (link).
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