Tagmentation and indexing of single cell suspensions (6x104 (link) cells) from three independent differentiations of WT (CCE) and Eomes-null (clone 6A6, CCE) day 4 Flk-1hi/PdgfRa- cells was performed as previously described38 (link). To control for sequence bias of the Tn5 transposase, 100ng genomic DNA of WT (CCE) ESCs was also tagmented and indexed. Libraries were purified with two rounds of Agencourt AMPure XP bead cleanup (Agencourt, 1.5× bead:sample ratio). Library size and concentration were determined using the 2100 Bioanalyzer High Sensitivity DNA Kit (Agilent). Samples were sequenced using a 75-cycle paired end Nextera kit with custom Nextera index primers taken from Table S1 in Buenrostro et al. 201338 (link) on the Illumina HiSeq4000 platform.
2100 bioanalyzer high sensitivity dna kit
Manufactured by Agilent Technologies
Sourced in United States
The 2100 Bioanalyzer High Sensitivity DNA Kit is a laboratory instrument used for the analysis of DNA samples. It provides high-sensitivity detection and sizing of DNA fragments ranging from 50 to 7,000 base pairs. The kit includes reagents, chips, and software to enable automated electrophoretic separation and fluorescent detection of DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Qubit dsdna high sensitivity kit, by Thermo Fisher Scientific (3 mentions)
Avenio cfdna isolation kit, by Roche (3 mentions)
Hiseq 2500, by Illumina (3 mentions)
Hiseq 4000, by Illumina (2 mentions)
Ampure xp bead cleanup, by Beckman Coulter (2 mentions)
Nextseq 500, by Illumina (2 mentions)
Hiseq 1000, by Illumina (2 mentions)
Nextseq 550, by Illumina (1 mentions)
Qubit high sensitivity dna kit, by Thermo Fisher Scientific (1 mentions)
Ion xpress barcode adaptors, by Thermo Fisher Scientific (1 mentions)
Rnadvance tissue kit, by Beckman Coulter (1 mentions)
Qiaseq library quant assay kit, by Qiagen (1 mentions)
Kingfisher flex platform, by Thermo Fisher Scientific (1 mentions)
Nebnext ultra 2 non directional rna second strand synthesis module, by New England Biolabs (1 mentions)
Nextseq 500 platform, by Illumina (1 mentions)
Nebnext ultra directional rna library prep kit, by New England Biolabs (1 mentions)
Nebnext rrna depletion kit, by New England Biolabs (1 mentions)
Nebnext multiplex oligos, by New England Biolabs (1 mentions)
Sensimix sybr, by Meridian Bioscience (1 mentions)
Kapa dna standards, by Illumina (1 mentions)
23 protocols using 2100 bioanalyzer high sensitivity dna kit
1
Single Cell Tagmentation and Sequencing
2
Small RNA Library Preparation for Illumina
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Small RNA libraries are prepared from 150 ng of total RNA using the Small RNA Library Prep Kit for Illumina (Norgen Biotek), according to the manufacturer’s instructions. Briefly, 3’ adapters were ligated to the RNA and excess adapters were removed using the included column-based cleanup. Following cleanup, 5’ adapters were ligated; the input RNA is now flanked by 3’ and 5’ adapters which were used to reverse transcribe the RNA. Following RT, unique indices were added through 14 rounds of PCR amplification. The final indexed PCR product was cleaned up using the included column-based cleanup. The eluate was run on a 6% TBE gel for size selection. The expected library size is ~140bp and the corresponding band was excised from the gel and columns purified. The final libraries were quantified using a Qubit DNA High Sensitivity kit (ThermoFisher) and the size was determined using a 2100 Bioanalyzer High Sensitivity DNA Kit (Agilent Technologies, CA, USA). Normalized libraries were pooled (48 samples per pool), denatured, diluted to 0.8 pmol/l, and loaded onto a High Output (75 cycle) flow cell (Illumina, CA, USA); followed by sequencing (1 × 75 bp) on a NextSeq 550 (Illumina).
3
Full Genome Sequencing of Archived Viruses
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All viruses taken from archived samples were subjected to full genome sequencing essentially as described before [44 (link)]. Briefly, RNA was automatically extracted on a KingFisher Flex platform (Thermo Fisher Scientific, Waltham, MA, USA) using the RNAdvance Tissue Kit (Beckmann Coulter, Brea, CA, USA). Double stranded cDNA was generated from 350 ng total RNA using the SuperScript IV First-Strand cDNA Synthesis System (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA) and the NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module (New England Biolabs, Ipswich, MA, USA). After conversion into cDNA, fragmentation was achieved by ultrasonication on a Covaris M220 (Covaris, Brighton, UK). Subsequently, Ion Torrent-specific sequencing libraries were generated using the GeneRead L Core Kit (Qiagen, Hilden, Germany) together with IonXpress barcode adaptors (Thermo Fisher Scientific). After quantification (QIAseq Library Quant Assay Kit, Qiagen) and quality control (2100 Bioanalyzer, High sensitivity DNA Kit, Agilent Technologies, Santa Clara, CA, USA) of the libraries, sequencing was performed on an Ion Torrent S5XL instrument utilizing Ion 530 chips and reagents according to the manufacturer’s instructions.
4
Genome-wide ChIP-seq and RNA-seq Protocols
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All cChIP–seq experiments were performed in biological triplicates. All ncRNA-seq experiments were performed in biological quadruplicates. Libraries for cChIP–seq and native cChIP–seq were prepared from 5–10 ng of ChIP and corresponding input DNA samples using a NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs), following the manufacturer’s guidelines. For ncRNA-seq, RNA samples (800 ng) were depleted of ribosomal RNA using the NEBNext rRNA Depletion kit (New England Biolabs). RNA-seq libraries were prepared using the NEBNext Ultra Directional RNA Library Prep kit (New England Biolabs). Samples were indexed using NEBNext Multiplex Oligos (New England Biolabs). The average size and concentration of all libraries were analyzed using the 2100 Bioanalyzer High Sensitivity DNA Kit (Agilent) followed by qPCR using SensiMix SYBR (Bioline, UK) and KAPA Illumina DNA standards (Roche). Libraries were sequenced as 40 bp paired-end reads on Illumina NextSeq 500 platform.
5
PacBio Iso-Seq Library Preparation
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SMRTbell library preparation of the pooled cDNA fractions used the SMRTbell Express Template Prep Kit 2.0 according to PacBio Iso-Seq protocol [PN 101-763-800 version 02 (October 2019)]. A fivefold dilution derived from the final SMRTbell library was used for assessing concentration and average library size by Qubit dsDNA HS and the 2100 Bioanalyzer High Sensitivity DNA Kit (Agilent), respectively. Volumetric calculations were done on a per-sample basis using SMRT Link Sample Setup for v4 primer and polymerase binding conditions dependent on initial SMRTbell library molarity. SMRTbell libraries were complexed with the Sequel II Binding Kit 2.0, purified with 1.0× ProNex beads, and sequenced at 60 pM on-plate loading concentration. Samples were sequenced on a single 8 M SMRT cell on the PacBio Sequel IIe platform using v2.0 chemistries. Sequencing included a 2-hour pre-extension and a 24-hour movie collection. The PacBio long-read RNA sequencing (Iso-Seq) data presented in this publication have been deposited in National Center for Biotechnology Information’s (NCBI’s) Sequence Read Archive and is available through accession number PRJNA752121.
6
Single Cell Tagmentation and Sequencing
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Tagmentation and indexing of single cell suspensions (6x104 (link) cells) from three independent differentiations of WT (CCE) and Eomes-null (clone 6A6, CCE) day 4 Flk-1hi/PdgfRa- cells was performed as previously described38 (link). To control for sequence bias of the Tn5 transposase, 100ng genomic DNA of WT (CCE) ESCs was also tagmented and indexed. Libraries were purified with two rounds of Agencourt AMPure XP bead cleanup (Agencourt, 1.5× bead:sample ratio). Library size and concentration were determined using the 2100 Bioanalyzer High Sensitivity DNA Kit (Agilent). Samples were sequenced using a 75-cycle paired end Nextera kit with custom Nextera index primers taken from Table S1 in Buenrostro et al. 201338 (link) on the Illumina HiSeq4000 platform.
7
Transcriptome Profiling by RNA-Seq
8
ScRNA-seq Library Preparation and Sequencing
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Sorted CD45– cells were converted to barcoded scRNA-seq libraries using Chromium Single Cell 3′ reagent kits (V3) (10X Genomics) according to the manufacturer’s instructions (CG000183 Rev A), aiming for 5,000–8,000 cells per library. Library quality control and quantification were performed using a KAPA Library Quantification kit for Illumina platforms (Kapa Biosystems, KK4873) and a 2100 Bioanalyzer High Sensitivity DNA kit (Agilent, 5067-4626). Libraries were sequenced on an Illumina HiSeq X Ten system with an average depth of 31,439 reads per cell, then mapped to the human genome (build GRCh38) and demultiplexed using CellRanger pipelines (10X Genomics, v.3.1.0).
9
RNA-seq of DIvA cells
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For total RNA-seq in DIvA cells, total RNA was isolated using the RNeasy Kit (Qiagen), according to the manufacturer's instructions. mRNA-seq indexed library preparation was performed starting from total mRNA (Illumina, TruSeq Stranded mRNA) according to the manufacturer's instructions. Library quality and quantity was assessed on the 2100 Bioanalyzer High Sensitivity DNA kit (Agilent), quantified on Qubit dsDNA HS Assay, normalized and pooled to perform a multiplexed sequencing run. Clusters were generated on the Illumina flow cell and sequencing was carried out on NextSeq 550 System with paired-end 75 bp. All six conditions were sequenced as biological triplicates. The reads for RNA-seq experiments were aligned to the GRCh37/hg19 assembly human reference genome using the STAR aligner [66] using default settings with the parameter --quantMode GeneCounts in order to obtain gene counts. Differential gene expression analysis was performed using the Bioconductor package DESeq2 (Love et al., 2014) that estimates variance-mean dependence in count data from high-throughput sequencing data and tests for differential expression exploiting a negative binomial distribution-based model.
10
Pacific Biosciences 10 kb SMRTbell Library Preparation
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DNA sequencing template was obtained from sheared genomic DNA using the Pacific Bioscience 10 kb SMRTbell library template preparation kit per the manufacturer's instructions (Pacific Biosciences, Menlo Park, CA, USA). The quality sizing analysis of DNA library was validated by Bioanalyzer 2100 high sensitivity DNA kit (Agilent Technologies, Inc., Santa Clara, CA, USA) prior to sequencing. PacBio RS II sequencing technology (Pacific Biosciences) was used as the sequencing platform. P4 chemistry was utilized, and the prepared library was sequenced on four single-molecule real-time (SMRT) cells.
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