Stably transduced iPSC‐MSCs were treated with or without doxycycline (50 ng/ml) for 24 h, then subjected to the ChIP‐seq process following the protocol reported previously [40 ]. Immunoprecipitation was performed using anti‐Flag M2 affinity gel (Sigma, A2220) according to the manufacturer's protocol. Eluted samples were incubated with RNase A (50 μg/ml final concentration) at 37 °C for 30 min, overnight with proteinase K to a final concentration of 200 μg/ml at 65 °C, and cleaned up using a Monarch PCR & DNA Cleanup Kit (T1030; New England BioLabs, Ipswich, MA, USA). Two biological replicates were prepared and sequenced independently. In brief, DNA was quantified using the Quant‐iT PicoGreen ds DNA assay (ThermoFisher, Waltham, MA, USA). Libraries were prepared with the HyperPrep Library Preparation Kit (PN 07962363001, Roche, Indianapolis, IN, USA). Libraries were quantified using an Quant‐iT PicoGreen ds DNA assay (ThermoFisher) or by low‐pass sequencing with a MiSeq nano kit (Illumina, San Diego, CA, USA). Single or paired end 50 cycle sequencing was performed on a NovaSeq 6000 platform (Illumina) at the Hartwell Center for Biotechnology at the St. Jude Children's Research Hospital. Details for ChIP‐seq data analysis are presented in Supplementary materials and methods.
Quant it picogreen dsdna assay
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom, Canada
The Quant-iT PicoGreen dsDNA assay is a fluorescence-based method for quantifying double-stranded DNA (dsDNA). It uses a dye that binds specifically to dsDNA, and the resulting fluorescence can be measured to determine the concentration of dsDNA in a sample.
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Lab products found in correlation
Nextera xt dna library preparation kit, by Illumina (15 mentions)
Nanodrop, by Thermo Fisher Scientific (14 mentions)
Novaseq 6000, by Illumina (12 mentions)
Ampure xp bead, by Beckman Coulter (11 mentions)
Agilent bioanalyzer dna 1000 kit, by Agilent Technologies (10 mentions)
Miseq nano kit, by Illumina (8 mentions)
Agencourt ampure xp bead, by Beckman Coulter (8 mentions)
Hiseq 4000, by Illumina (7 mentions)
2100 bioanalyzer high sensitivity kit, by Agilent Technologies (7 mentions)
Papain, by Merck Group (7 mentions)
Agencourt ampure xp system, by Beckman Coulter (7 mentions)
Truseq stranded total rna library prep kit, by Illumina (6 mentions)
2100 bioanalyzer rna 6000 nano assay, by Agilent Technologies (6 mentions)
Sytox green, by Thermo Fisher Scientific (6 mentions)
Hiseq 2500, by Illumina (6 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Agilent bioanalyzer high sensitivity assay, by Agilent Technologies (5 mentions)
Nextseq, by Illumina (5 mentions)
Nextseq high output platform, by Illumina (5 mentions)
Qiacube, by Qiagen (5 mentions)
253 protocols using quant it picogreen dsdna assay
1
Chromatin Immunoprecipitation Sequencing of iPSC-MSCs
2
Bisulfite Sequencing of DNA Methylation
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Library preparation and sequencing were carried out at NU-OMICS DNA Sequencing research facility (Northumbria University). Purified PCR products were quantified using Quant-iT™ PicoGreen™ dsDNA Assay (Invitrogen) and normalized to 20 nM and pooled by experimental condition. Libraries of pooled amplicons were prepared using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (New England Biolabs). The quality of prepared libraries was assessed using the Agilent High Sensitivity DNA Kit Guide (Agilent) and quantified using Quant-iT™ PicoGreen™ dsDNA Assay (Invitrogen). Libraries were normalized to 4 nM, pooled, and sequenced using the Illumina MiSeq V2 300 cycle chemistry. Fastq files containing sequencing data were uploaded to EPIC TABSTAT v1.7 (https://tabsat.ait.ac.at/ ) for analysis in paired-end mode with mouse mm10 genome used as the reference and Bowtie2 as the mapping tool. The Bismark output files produced % methylation at each CpG site sequenced for each sample.
3
Whole Genome Amplification by MDA
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DNA was denatured by mixing the DNA diluted in milliQ water 1:1 with 50 mM KOH (Sigma Aldrich) and incubating for 3 min at room temperature (RT). The denatured DNA was the neutralized by adding an equal volume of Tris-HCl (80 mM, pH4; Sigma Aldrich). RepliPHI Phi29 Reagent Kit (Epicenter) supplemented with Exo-Resistant Random Primer (ThermoFisher Scientific) was used for the MDA reaction. A 2× MDA mastermix (2× reaction buffer, 2 mM dNTP, 50 μM primer, 4 U/μl Phi29, 8 mM DTT and 5 % DMSO) was prepared. The denatured and neutralized DNA and the 2× MDA mastermix were mixed at equal volumes by pipetting for a bulk reaction in tube or in the microfluidic chip as described above for emulsion generation. Reactions were incubated for 12 h at 30 °C. The polymerase was then inactivated at 65 °C for 10 min.
After incubation, the emulsion was broken by adding 5 μl 1H, 1H, 2H, 2H, Perfluoro-1-octanol (Sigma Aldrich), vortexing, and centrifuging briefly until the emulsion separated into one aqueous and one oil phase. If the emulsion did not break, the emulsion breaking procedure was repeated. The supernatant (aqueous phase) was collected by pipetting and could then be treated like the MDA products from the bulk reactions. The concentrations of MDA products were quantified with Qubit dsDNA kit (ThermoFisher Scientific) or Quant-iT PicoGreen dsDNA assay (ThermoFisher Scientific).
After incubation, the emulsion was broken by adding 5 μl 1H, 1H, 2H, 2H, Perfluoro-1-octanol (Sigma Aldrich), vortexing, and centrifuging briefly until the emulsion separated into one aqueous and one oil phase. If the emulsion did not break, the emulsion breaking procedure was repeated. The supernatant (aqueous phase) was collected by pipetting and could then be treated like the MDA products from the bulk reactions. The concentrations of MDA products were quantified with Qubit dsDNA kit (ThermoFisher Scientific) or Quant-iT PicoGreen dsDNA assay (ThermoFisher Scientific).
4
Chromatin Sonication and Gel Extraction
5
454 Sequencing of 16S rRNA Gene
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For 454 sequencing the FAP64 primer was fused to the 454 Lib-L Adaptor A and one of 20 different 10 base pair multiplex identifiers (MIDs) [18] (link). The FAP59 primer was fused to the 454 Lib-L Adaptor B. PCR was performed as described above, except the cycling conditions were 95 °C for 2 min followed by 32 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min. Amplicons were gel purified using the MinElute gel extraction kit (Qiagen) and quantified using the Quant-iT Picogreen dsDNA assay (ThermoFischer). Amplicons were pooled in equal amounts with 20 amplicons per library. Sequencing was done by Macrogen (Seoul, South Korea) on the 454 GS-FLX platform using Titanium chemistry on 1/8 region of the plate per library.
6
Extraction and Sequencing of Mycobacterial DNA
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Samples were cultured once on Middlebrook 7H10 agar and plate sweeps were collected for DNA extraction using the van Soolingen method (van Soolingen et al., 1991 (link)). Genomic DNA was quantified using the Quant-iT PicoGreen dsDNA Assay (ThermoFisher Scientific, Massachusetts, USA). Library preparation and sequencing were done at the McGill University/Genome Québec Innovation Centre. The Illumina HiSeq 4000 was used to produce paired-end 100 bp reads. To obtain the depth of coverage needed for this study (~500–1000x for deep sequencing, compared to ~50–100x as routinely done by public health), pooled libraries were run on four independent lanes.
7
PCR-Free DNA Quantification and Normalization
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The following MlyI digests were set up for PCR-free quantification: 200–500 ng even or staggered pool DNA, 2 μl Cutsmart buffer (NEB), 1 μl MlyI (NEB), and volume was adjusted to 20 μl with nuclease-free water. Digests were incubated at 37 °C for 1 h, followed by 20 min at 65 °C. Thirty microliters of water was added to each digest (to bring the volume up to 50 μl). Thirty microliters (0.6×) of AmpureXP beads (Beckman Coulter) was added, and after a 5-min incubation, beads were collected on a magnet and the supernatant was transferred to a new tube (discarded beads). Eighty microliters (1×) of AmpureXP beads was added, the beads were washed two times for 30 s using fresh 80% ethanol, and the beads were air dried for 10 min, followed by elution in 20 μl of EB (Qiagen). Libraries were quantified using a Quant-iT PicoGreen dsDNA assay (Thermo Fisher Scientific), fragment sizes were assessed using an Agilent Bioanalyzer High Sensitivity assay, and libraries were normalized to 2 nM for sequencing.
8
Methylome Sequencing Protocol with Controls
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DNA for methylome sequencing was extracted using the Qiagen DNEasy Plant 96 kit in accordance with the manufacturer’s instructions, and checked for intactness on a 1% agarose gel and quantitated using the Thermo Fisher Quant-iT PicoGreen dsDNA Assay. Of Hedin/2 genomic DNA, 200 ng was combined with 0.001 ng of CpG-methylated pUC19 control DNA and 0.02 ng of unmethylated bacteriophage Lambda control DNA, then brought to a volume of 50 µl using EB buffer. The input DNA was sheared to 350–400 bp on the S220 Focused-Ultrasonicator Instrument (Covaris) using the following protocol: duty factor = 10; peak incident power = 175; cycles per burst = 200; time = 2 times 30 s. The sheared DNA was used to prepare a large insert NEBnext Enzymatic Methyl-seq library following the manufacturer’s instructions (https://www.neb.com/-/media/nebus/files/manuals/manuale7120.pdf ). Four libraries were constructed with different sequencing indexes. Index PCR was performed with five PCR cycles to include indexes and amplify the libraries. The final libraries were quantified by quantitative PCR, pooled at equimolar concentrations and sequenced for 500 cycles (2 × 250 bp paired-end reads) on an SP-flow cell of the Novaseq6000 system (Illumina).
9
Quantification of DNA Removal in Decellularized Heart Tissue
10
Amplification and Sequencing of Bacterial V4 16S rRNA
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The V4 hypervariable region of the 16S rRNA gene was amplified using a universal forward sequencing primer and a uniquely barcoded reverse sequencing primer to allow for multiplexing [27 (link)]. Amplification reactions were performed using 12.5 μl of KAPA2G Robust HotStart ReadyMix (KAPA Biosystems), 1.5 μl of 10 μm forward and reverse primers, 8 μl of sterile water, and 1.5 μl of DNA. The V4 region was amplified by cycling the reaction at 95 °C for 3 min, 30× cycles of 95 °C for 15 s, 50 °C for 15 s, and 72 °C for 15 s, followed by a 5-min 72 °C extension. All amplification reactions were done in triplicate, checked on a 1% agarose TBE gel, and then pooled to reduce amplification bias. Pooled triplicates were quantified using Quant-it PicoGreen dsDNA Assay (Thermo Fisher Scientific) and combined by even concentrations. The final library was purified using Ampure XP beads (Agencourt), selecting for the bacterial V4 amplified band. The purified library was quantified using Qubit dsDNA Assay (Thermo Fisher Scientific) and loaded on to the Illumina MiSeq for sequencing, according to manufacturer instructions (Illumina, San Diego, CA, USA). Sequencing was performed using the V2 (150 bp × 2) chemistry. Sequencing depths are reported in Additional file 3 .
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