For 375 of the 576 patients complete clinical information as well as genetic information from a 36-gene panel targeted next-generation sequencing assay were available for prognostic modeling. The regions of these 36 genes were selected for custom target capture using Agilent SureSelect Target Enrichment Kit (Agilent Technologies, Santa Clara, United States). Libraries derived from each DNA sample were prepared using NEB Ultra II (New England Biolabs, Ipswitch, United States) and individually barcoded by dual indexing. Sequencing was performed on an HiSeq 4000 (Illumina) with 150 bp paired-end reads. Forty-eight pooled libraries per lane were sequenced to a median read depth of ~400x. The custom panel of target regions covered all coding regions and consensus splice sites from the following 36 genes: ASXL1, CALR, CBL, CEBPA, DNMT3A, EZH2, FLT3, IDH1, IDH2, IKZF1, JAK2, KRAS, MPL, NPM1, NRAS, PHF6, PTPN11, RUNX1, SETBP1, SF3B1, SH2B3, SRSF2, TET2, TP53, U2AF1, and ZRSR2. Paired-end reads were processed and analyzed as previously described8 (link).
Neb ultra 2
Manufactured by New England Biolabs
Sourced in United States
NEB Ultra II is a high-performance DNA polymerase designed for a wide range of PCR applications. It provides robust amplification with high fidelity and sensitivity.
Automatically generated - may contain errors
Lab products found in correlation
Rnaqeous micro, by Thermo Fisher Scientific (2 mentions)
Agilent sureselect target enrichment kit, by Agilent Technologies (1 mentions)
Hiseq 4000, by Illumina (1 mentions)
Hiseq x, by Illumina (1 mentions)
Agilent d1000 tape station, by Agilent Technologies (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
S220 focused ultrasonicator, by Covaris (1 mentions)
Neb lunascript rt supermix kit, by New England Biolabs (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
6 protocols using neb ultra 2
1
Targeted NGS for Prognostic Modeling
2
RNA-seq of Memory CD4+ T Cells
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Memory CD4+ T cells, including from BACH2WT/L24P patients, were activated with VitD or carrier. 6x105 cells were pelleted at 300g for 5 min and RNA extracted by RNAqeous Micro (Thermo Fisher Scientific). 1μg total RNA for each sample was subjected to NEBNext Poly(A) mRNA Magnetic Isolation (E7490) and resulting mRNA prepared for RNA-seq by NEB Ultra II (New England Biolabs) and sequenced by HiSeq (Illumina).
The expression levels of all genes in RNA-seq libraries were quantified by ‘rsem-calculate-expression’ in RSEM/v1.3.162 (link) with parameters ‘--bowtie-n 1 --bowtie-m 100 --seed-length 28 --bowtie-chunkmbs 1000’. The bowtie index for RSEM alignment was generated by ‘rsem-prepare-reference’ on all RefSeq genes, downloaded from UCSC table browser in April 2017. EdgeR/v3.26.863 (link) was used to normalize gene expression among all libraries and identify DEGs among samples. Microarray analysis sourced from GSE119416 was carried out using Partek Genomics Suite (Partek, Inc.). DEGs were defined using the following criteria: at least 1.5-fold change in either direction at p-value<0.05 for microarray; at least 1.75-fold change in either direction at FDR<0.05 for RNA-seq.
The expression levels of all genes in RNA-seq libraries were quantified by ‘rsem-calculate-expression’ in RSEM/v1.3.162 (link) with parameters ‘--bowtie-n 1 --bowtie-m 100 --seed-length 28 --bowtie-chunkmbs 1000’. The bowtie index for RSEM alignment was generated by ‘rsem-prepare-reference’ on all RefSeq genes, downloaded from UCSC table browser in April 2017. EdgeR/v3.26.863 (link) was used to normalize gene expression among all libraries and identify DEGs among samples. Microarray analysis sourced from GSE119416 was carried out using Partek Genomics Suite (Partek, Inc.). DEGs were defined using the following criteria: at least 1.5-fold change in either direction at p-value<0.05 for microarray; at least 1.75-fold change in either direction at FDR<0.05 for RNA-seq.
3
ChIP-Seq Library Preparation with NEB Ultra II
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ChIP libraries were prepared using NEB Ultra II (Cat # E7103L), following NEBNext protocols with minor modifications. To reduce biases induced by PCR amplification of a repetitive region, libraries were prepared from 80–100 ng of input or ChIP DNA. The DNA was end-repaired and A-tailed, and NEBNext adapters (Cat # E7335S) were ligated. The libraries were PCR-amplified using only five to seven PCR cycles because the starting DNA amount was high. Libraries were run on a 2% agarose gel and size selected for 200–350 bp. Resulting libraries were sequenced using 150 bp, paired-end sequencing on a HiSeq X instrument per the manufacturer’s instructions (Illumina).
4
RNA-seq of Memory CD4+ T Cells
5
Optimized Sonication and Library Preparation for 3C Experiments
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Sonication of 3C libraries was performed using 3–5 μg per library on a Covaris S220 Focused Ultrasonicator with the following conditions: 250–300 s: duty cycle 10%; intensity 5; cycles per burst 200, to yield an average fragment size of 200 bp. The sonication quality was assessed using the Agilent D1000 TapeStation. The DNA was purified using Ampure XP beads (Beckman Coulter, A63881). To maximize library complexity, the addition of sequencing adapters was parallelized in triplicate reactions such that each reaction contained 1–2 μg of sonicated 3C library. NEB Ultra II (NEB, 7645 S) reagents were used following the manufacturer’s protocol with the following deviations: (1) 2–3× the number of adapters was used; (2) all Ampure XP bead clean-up reactions were performed with a DNA sample:bead ratio of 1:1.5; (3) to maximize library complexity and yield, the PCR was performed in triplicate per ligation reaction using the Herculase II PCR reagents (Agilent Technologies, 600677). The parallel library preparations and PCR reactions were subsequently pooled for each reaction.
6
Wastewater SARS-CoV-2 Genomic Surveillance
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RNA extracts from wastewater samples were used to produce amplicons and to prepare libraries according to the COVID-19 ARTIC v3 protocol24 with minor modifications. Briefly, extracted RNA was reverse transcribed using the NEB LunaScript RT SuperMix kit (New England Biolabs) and the resulting complementary DNA was amplified with the ARTIC v3 panel from IDT. ARTIC primers used were: ARTIC V4.1 NCOV-2019 Panel, 500rxn 10011442, IDT ARTIC v3 panel 500rxns 10006788 (IDT). The amplicons were end-repaired and polyadenylated before ligation of adapters using NEB Ultra II (New England Biolabs). Fragments containing adapters on both ends were selectively enriched and barcoded with unique dual indexing with PCR. Libraries were sequenced using the Illumina NovaSeq 6000 and MiSeq platforms, resulting in paired-end reads of 250 bp length each (see Supplementary Information for quality metrics of the sequencing data).
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