ATAC-seq was performed as described previously (44 (link)). For each cell line, 50,000 cells were harvested from 3 separate cultures and used to prepare tagmented chromatin (3 replicates of WT and 3 replicates of MGAT5 KO cell lines, 6 samples total). Quality of PCR-amplified sequencing libraries was assessed using a Tapestation 2200 instrument with high sensitivity DNA screentapes (Agilent). Libraries were sequenced as paired end reads on a single lane of an Illumina HiSeq4000 flow cell. Resulting reads were aligned to the GRCh37/hg19 reference genome using Rsubread (45 (link)), and alignments were filtered to remove low quality, duplicate, and mitochondrial reads. Peaks were called using MACS2 (46 ) on merged reads from all samples, and differential peak accessibility between cell lines was determined using edgeR (47 (link)) with a threshold false discovery rate of 5%. Transcription factor binding motifs enriched in differentially accessible peaks were identified using HOMER (48 (link)). H3K4me3 ChIP-seq data were downloaded from ENCODE1 and are available under accession ENCFF756EHF .
High sensitivity dna screentape
Manufactured by Agilent Technologies
Sourced in United States
The High Sensitivity DNA ScreenTape is a lab equipment product from Agilent Technologies. It is designed for the rapid and sensitive analysis of DNA samples using the Agilent 2200 TapeStation system.
Automatically generated - may contain errors
Lab products found in correlation
Tapestation 4200, by Agilent Technologies (3 mentions)
Kapa rna hyperprep kit with riboerase hmr, by Roche (3 mentions)
Kapa duel indexed adapters, by Roche (3 mentions)
Hiseq 4000 platform, by Illumina (3 mentions)
Rnaeasy mini kit, by Qiagen (3 mentions)
Nextseq 500, by Illumina (3 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (3 mentions)
Agilent high sensitivity rna screentape, by Agilent Technologies (3 mentions)
Ercc exfold spike in mixes, by Thermo Fisher Scientific (3 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Kapa library quantification kit, by Roche (2 mentions)
Lightcycler 480, by Roche (2 mentions)
Truseq stranded mrna library prep kit, by Illumina (2 mentions)
Truseq stranded mrna library preparation kit, by Illumina (2 mentions)
Tapestation 2200, by Agilent Technologies (2 mentions)
Iscript advanced cdna synthesis kit, by Bio-Rad (2 mentions)
Ddpcr evagreen supermix kit, by Bio-Rad (2 mentions)
Qx200 droplet reader, by Bio-Rad (2 mentions)
Quantasoft software, by Bio-Rad (2 mentions)
2200 tapestation instrument, by Agilent Technologies (1 mentions)
13 protocols using high sensitivity dna screentape
1
Profiling Chromatin Accessibility via ATAC-seq
2
RNA-seq library preparation from degraded samples
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In collaboration with the Wales Gene Park (Cardiff University), 5 ng of total RNA was depleted of ribosomal RNA using the NEBNext® rRNA Depletion Kit (Human/Mouse/Rat), (New England BioLabs, (UK) Ltd). Ribosomal RNA depletion of each sample was assessed using the Agilent 4200 TapeStation with hsRNA ScreenTape (Agilent Technologies, Inc, UK).
The sequencing libraries were prepared using the NEB® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England BioLabs, (UK) Ltd) protocol. The steps included RNA fragmentation and priming, 1 st strand cDNA synthesis, 2 nd strand cDNA synthesis, adenylation of 3' ends, adapter ligation (adapter diluted 1:199) and PCR ampli cation (16-cycles). Following the addition of the PCR enrichment master mix, a unique index primer was added to each sample. The standard fragmentation procedure of 15 min at 94°C for samples for intact RNA (>7) was reduced to 8 min at 94°C for partially degraded RNA with RIN values 2-6. Except for the replacement of RNAClean ® XP beads and SPRI select beads by AMPure XP beads (Beckman Coulter ® ) the manufacturer's instructions were followed. The libraries were validated using Agilent 4200 TapeStation and high sensitivity DNA ScreenTapes (Agilent Technologies, Inc, UK) to ascertain the insert size, and the Qubit® (Thermo Fisher Technologies, UK) was used to perform the uorometric quantitation.
The sequencing libraries were prepared using the NEB® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England BioLabs, (UK) Ltd) protocol. The steps included RNA fragmentation and priming, 1 st strand cDNA synthesis, 2 nd strand cDNA synthesis, adenylation of 3' ends, adapter ligation (adapter diluted 1:199) and PCR ampli cation (16-cycles). Following the addition of the PCR enrichment master mix, a unique index primer was added to each sample. The standard fragmentation procedure of 15 min at 94°C for samples for intact RNA (>7) was reduced to 8 min at 94°C for partially degraded RNA with RIN values 2-6. Except for the replacement of RNAClean ® XP beads and SPRI select beads by AMPure XP beads (Beckman Coulter ® ) the manufacturer's instructions were followed. The libraries were validated using Agilent 4200 TapeStation and high sensitivity DNA ScreenTapes (Agilent Technologies, Inc, UK) to ascertain the insert size, and the Qubit® (Thermo Fisher Technologies, UK) was used to perform the uorometric quantitation.
3
Viral Whole-Genome Sequencing for SARS-CoV-2
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The viral whole-genome sequencing was performed on extracted RNA from the isolated virus. Briefly, this protocol [36 (link),37 (link)] consisted of: reverse-transcription with the SuperScriptTM VILOTM cDNA synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA); PCR enrichment of the SARS-CoV-2 genome and five human gene expression controls with the Ion AmpliSeqTM SARS-CoV-2 Research Panel; library construction with the Ion AmpliSeqTM Library Kit Plus; library quantification and size range verification at the 2200 TapeStation Automated Electrophoresis System, using the High Sensitivity DNA ScreenTape (Agilent Technologies, Santa Clara, CA, USA); next-generation sequencing (NGS) on the Ion S5XL system with the Ion 530™ chip; and raw data extracted with the Ion Torrent pipeline.
The bioinformatic pipeline consisted of: alignment of the raw data versus the reference genome (severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1; accession number NC_045512.2; [38 (link)]) with BWA tool; variant calling with three tools, FreeBayes, BCFtools and GATK, with editing of variants identified in at least two; variant annotation with SnpEff; consensus sequence was inferred with Bcftools.
The bioinformatic pipeline consisted of: alignment of the raw data versus the reference genome (severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1; accession number NC_045512.2; [38 (link)]) with BWA tool; variant calling with three tools, FreeBayes, BCFtools and GATK, with editing of variants identified in at least two; variant annotation with SnpEff; consensus sequence was inferred with Bcftools.
4
RNA-seq of sorted neurons and whole worms
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For RNA-seq of samples from sorted neurons (the sort group) and whole worms (the whole group), libraries were prepared using KAPA RNA HyperPrep kit with RiboErase (HMR) (KAPA biosystems) according to the manufacturer’s protocol. The RNA input was 150 ng and fragmentation conditions were 85oC for 5 min. Barcodes were introduced to each sample using KAPA duel-indexed adapters (KAPA biosystems). Length distribution of each library was determined by TapeStation 4200 (Agilent) using High Sensitivity DNA ScreenTape (Agilent). Libraries were quantified by KAPA library quantification kit (KAPA biosystems) and then multiplexed and sequenced on Illumina Hiseq 4000 platform to obtain 150 nt paired-end reads.
5
ChIP-seq for Histone Modifications
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The ChIP-seq protocol was adapted from Schmidt and colleagues (Schmidt et al., 2009 (link)) and carried out from OSCs according to Fabry and colleagues (Fabry et al., 2019 (link)) using commercially available rabbit polyclonal anti-H3K9me3 (Active Motif, cat # 39161) and rabbit polyclonal anti-H3K4me2 (Merck Millipore, cat # 07–030) antibodies. ChIP from S2 cells was carried out using the same protocol from 2 × 107 cells with a modification to the chromatin preparation procedure to sonicate chromatin for 8 cycles of 30 s on/30 s off using a Bioruptor Pico (Diagenode). Recovered DNA was quantified on an Agilent TapeStation System using a High Sensitivity DNA ScreenTape (Agilent). DNA sequencing libraries were prepared with NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB; E7645) according to the manufacturer’s instructions and quantified with KAPA Library Quantification Kit for Illumina (Kapa Biosystems). OSC and S2 cell ChIP-seq libraries were sequenced on an Illumina HiSeq 4000 and NovaSeq 6000, respectively.
6
RNA-seq of RA/SAG Differentiated Cells
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Cells were collected before TF induction (day 2 of RA/SAG differentiation) and 48 h after Dox treatment (day 4 of RA/SAG differentiation). RNA was extracted by using TRIzol LS Reagent (Life Technologies) and purified using the RNAeasy Mini Kit (Qiagen). Agilent High Sensitivity RNA Screentape (Agilent, 5067-5579) was used to check RNA integrity. A 500 ng quantity of RNA was used to prepare RNA-seq libraries and spiked-in with ERCC ExFold Spike-In mixes (Thermo Fisher Scientific, 4456739). RNA-seq libraries were prepared using a TruSeq Stranded mRNA Library Prep kit (Illumina, 20020594). Library size was verified using High Sensitivity DNA ScreenTape (Agilent, 5067-5584). The KAPA Library Amplification kit was used on a Roche LightCycler 480 for library quantification before pooling. The libraries were sequenced on an Illumina NextSeq 500 using V2.5 chemistry (75 cycles, single-end 75 bp) at the Genomics Core Facility at New York University.
7
RNA-seq of cells grown in +2i/-2i
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Cells were collected after grown for 3d in +2i or −2i conditions in the absence of doxycycline. TRIzol LS Reagent (LifeTechnologies) was used to extract RNA and RNAeasy mini kit (Qiagen) used to purify RNA, as in (Bulajić et al., 2020 (link)). Agilent High Sensitivity RNA Screentape (Agilent) was used to determine RNA integrity. 500 ng RNA from cells that was spiked-in with ERCC Exfold Spike-in mixes (Thermo Fisher, 4456739) was used for the generation of RNA-seq libraries. TruSeq Stranded mRNA Library Preparation kit (Illumina, 20020594) was used to prepare RNA-seq libraries. High Sensitivity DNA ScreenTape (Agilent, 5067-5584) was used to verify library sizes. A KAPA library amplification kit was used on Roche Lightcycler 480 to quantify the library prior to sequencing. Libraries were sequenced on Illumina NextSeq 500 using V2.5 chemistry (75 cycles, single-end 75bp) at the Genomics Core Facility at NYU.
8
RNA-seq of cells grown in +2i/-2i
9
Profiling miRNA in Asthmatic Adipose Tissue
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RNA and miRNA from epididymal adipose tissue (six asthmatic and sic control rats) were extracted using ExtractMe miRNA kit (Blirt, Gdansk, Poland). The quantity of miRNA samples was measured using microRNA assay kit (ThermoFisher Scientific, Foster City, CA, USA). The miRNA expression profile from epididymal adipose tissue and lung tissue was performed using next-generation sequencing. Libraries were generated from 50 ng of miRNA sample using TruSeq small RNA library preparation kit (Illumina, San Diego, CA, USA) following the manufacturer’s instructions. After size selection in polyacrylamide 8% gel electrophoresis, libraries were validated and quantified using High Sensitivity DNA Screen Tape (Agilent, Santa Clara, CA, USA) on Tape Station 2200 (Agilent) and run on MiniSeq sequencer (Illumina, San Diego, CA, USA) with 50 nt single-end reads. Differential miRNA expression analysis was done in Base Space software (DeSeq algorithm, Illumina, San Diego, CA, USA), and reads were mapped to the rat genome (miRbase v21). Raw sequencing data were stored in NCBI SRA database (http://www.ncbi.nlm.nih.gov/bioproject/657964 ).
10
Bulk RNA-seq of Sympathetic and Hippocampal Neurons
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We initiated bulk RNA-seq experiments on sympathetic and hippocampal neurons that are grown under young and mature culture conditions (Figures 1 , 8 ). The SCG is harvested from rat pups of two pregnant rats, dissociated and 100,000 cells plated in 12-well plate. Hippocampal cultures were made from E18 embryos, dissociated and 500.000 cells plated on six well plates. Cells are treated with antimitotic agents for 5 days and total RNA is harvested using Trizol. RNA extractions were quantified using RNA Nano Chips (Cat. #5067–1511) on an Agilent 2100 BioAnalyzer. RNA-Seq library preps were constructed using the Illumina TruSeq Stranded mRNA Library Prep kit (Cat #20020595) using 500 ng of total RNA as input, amplified by 11 cycles of PCR. Final libraries were visualized using High Sensitivity DNA ScreenTape (Agilent, Cat. #5067–5584) on the Agilent TapeStation 2200 instrument. Quant-It (Invitrogen, Cat. P11495) was used for final concentration determination and libraries were pooled equimolar. The pool was sequenced paired-end 50 cycles on an Illumina NovaSeq6000 SP 100 Cycle flowcell-v1.5 with 2% PhiX spike-in.
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