RNA-seq was performed as previously outlined [50 ] using Nextera transposases (Illumina). Briefly, cells were lysed with Buffer RLT (Qiagen) containing 10 % beta-mercaptoethanol. The Norgen Animal Tissue RNA Purification Kit (Norgen Biotek) was used to isolate RNA from cells. The Dynabead mRNA Purification Kit (Invitrogen) beads were used to isolate mRNA transcripts and cDNA synthesis was performed using Superscript reverse transcriptase (Invitrogen). Nextera transposases (Illumina) were used to fragment cDNA prior to next-generation sequencing. All experimental treatments at all time points were performed in quadruplicate.
Nextera transposase
Manufactured by Illumina
The Nextera transposase is a core component of the Nextera library preparation workflow. It is an enzymatic tool used to simultaneously fragment and tag DNA samples, preparing them for next-generation sequencing. The Nextera transposase enables rapid, efficient, and streamlined library construction.
Automatically generated - may contain errors
Lab products found in correlation
Qg buffer, by Qiagen (3 mentions)
Agencourt ampure xp bead, by Beckman Coulter (3 mentions)
Superscript vilo cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
Dna polymerase 1, by New England Biolabs (3 mentions)
Hiseq 2000, by Illumina (3 mentions)
Dynabead, by Thermo Fisher Scientific (2 mentions)
Hiseq 2000 instrument, by Illumina (2 mentions)
Protein g dynabead, by Thermo Fisher Scientific (2 mentions)
Nextseq 500, by Illumina (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Superscript reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Dynabead mrna purification kit, by Thermo Fisher Scientific (1 mentions)
Norgen animal tissue rna purification kit, by Norgen Biotek (1 mentions)
Nextera tagmentation, by Illumina (1 mentions)
Streptavidin dynabeads, by Thermo Fisher Scientific (1 mentions)
Ampure xp beads, by Qiagen (1 mentions)
9 protocols using nextera transposase
1
RNA-seq Transcriptome Profiling Protocol
2
RNA-seq Library Construction and Analysis
3
ATAC-seq Analysis of Mouse Cell Lines
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Assay for transposase-accessible chromatin with high-throughput sequencing analysis was performed as described [33 (link)]. Briefly, 50,000 nuclei from αT1–1, αT3–1 and LβT2 cell lines were transposed using Illumina Nextera Transposase. Library fragments were amplified using NEBnext PCR master mix and custom Nextera PCR primers. Sequencing was performed on a NextSeq 500 system at the ICM iGenSeq core facility from Paris. Three independent replicates were done for each line. Data analysis was performed according to Buenrostro et al. [33 (link)]. Briefly, ATAC-seq reads were mapped on mouse genome mm9 using Bowtie 2 [34 (link)] and peak calling was done using MACS2 [35 (link)]. Peaks were then tested for consistency among three independent replicates, and data visualization was done using IVG software [36 (link)].
4
ChIA-PET Protocol with Chromatin Shearing
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We performed ChIA-PET experiments with modifications to previously published protocols2 (link),22 (link). These modifications have also been independently described17 (link),23 (link). We used Illumina’s Nextera tagmentation to generate sequencing libraries. In brief, cells were crosslinked and subjected to nuclear lysis followed by chromatin shearing (no restriction enzyme was used). Immunoprecipitation was performed overnight at 4 °C with antibodies against the cohesin subunit RAD21 (Abcam Anti-RAD21 antibody (ab992) https://www.encodeproject.org/antibodies/ENCAB529YRC/ ). The immuno-complexes were pulled down with Protein-G dynabeads (Life Technologies #10003D, New York). Biotinylated linkers were ligated to the enriched fragments, followed by proximity ligation overnight at 16 °C.
Crosslinking was reversed at 65 °C with the use of Proteinase K followed by DNA purification. We used Illumina Nextera Transposase to add sequencing adapters to ChIA-PET libraries. Biotinylated fragments were enriched by pull-down with Streptavidin Dynabeads (M-280; Lifetechnologies #11205D, New York). The final libraries were sequenced on an Illumina HiSeq 2000.
Crosslinking was reversed at 65 °C with the use of Proteinase K followed by DNA purification. We used Illumina Nextera Transposase to add sequencing adapters to ChIA-PET libraries. Biotinylated fragments were enriched by pull-down with Streptavidin Dynabeads (M-280; Lifetechnologies #11205D, New York). The final libraries were sequenced on an Illumina HiSeq 2000.
5
RNA-seq Library Construction and Analysis
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RNA-seq libraries were constructed as previously described46 . Briefly, seven days after transduction, cells were harvested and mRNA was purified from total RNA using oligo(dT) Dynabeads (Invitrogen). First-strand cDNA was synthesized using the SuperScript VILO cDNA Synthesis Kit (Invitrogen) and second-strand cDNA was synthesized using DNA polymerase I (New England Biolabs). cDNA was purified using Agencourt AMPure XP beads (Beckman Coulter). Purified cDNA was treated with Nextera transposase (Illumina) for 5 min at 55 °C to simultaneously fragment and insert sequencing primers into the double-stranded cDNA. Transposase activity was halted using QG buffer (Qiagen) and fragmented cDNA was purified on AMPure XP beads. Indexed sequencing libraries were PCR-amplified and sequenced for 50-bp paired-end reads on an Illumina HiSeq 2000 instrument at the Duke Genome Sequencing Shared Resource and for 75 paired-end reads on an Illumina MiSeq. Reads were trimmed to 50 bp and aligned to the delivered lentiviral vector were removed from analysis using Bowtie247 . Filtered reads were then aligned to human RefSeq transcripts using Bowtie2. Statistical analysis, including multiple hypothesis testing, on three independent biological replicates was performed using DESeq48 .
6
ATAC-Seq Preparation Protocol
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Samples were prepared for ATAC-seq essentially as described previously (Buenrostro et al., 2013 (link)). Macrophages were washed twice in cold PBS, scraped in cold PBS, and counted using a hemocytometer. Based on this count, 50,000 cells were aliquoted and pelleted by centrifugation. Cell pellets were washed once with 50 μL cold PBS on ice before lysis in 50 μL hypotonic lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630) over the course of a 10 minute spin at 4°C. Pelleted nuclei were resuspended in 50 uL transposition reaction mix with 3 uL Nextera transposase per sample (Illumina). The reaction was stopped with 0.1% SDS and transposase-accessible DNA was isolated using AMPure XP beads (Beckman-Coulter). Accessible DNA was amplified by PCR for 5 cycles, assessed for yield by qPCR, and amplified for an additional 7 cycles. Libraries were sequenced on a NextSeq 500 (Illumina).
7
CTCF-mediated Chromatin Interactions by ChIA-PET
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We performed ChIA-PET experiments with modifications to previously published protocols33 ,34 (link) and as described in the following: Cells were crosslinked and subjected to nuclear lysis followed by chromatin shearing. Immunoprecipitation was performed overnight at 4 °C with antibodies against CTCF (Cell signaling technology [#3418]). The immuno-complexes were pulled down with Protein-G dynabeads (Life Technologies #10003D, New York). Biotinylated linkers were ligated to the enriched fragments, followed by proximity ligation overnight at 16 °C. Crosslinking was reversed at 65 °C with use of Proteinase K followed by DNA purification. We used Illumina Nextera Transposase to add sequencing adapters to ChIA-PET libraries, thus using Illumina’s Nextera tagmentation to generate sequencing libraries, which was different from the previously published protocols33 ,34 (link). Biotinylated fragments were enriched by pull-down with Streptavidin Dynabeads (M-280; Lifetechnologies #11205D, New York). The final libraries were sequenced on the Illumina HiSeq 2000. The data was processed following the Mango toolkit35 (link).
8
ATAC-Seq Preparation Protocol
9
RNA-seq Library Preparation and Analysis
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