BJ cells (passage 19) were harvested at 70% confluence using Accutase and washed twice with ice-cold PBS. Aliquots (8,000 cells) were pelleted for 5 min at 500g in 1.5 mL tubes at 4 °C, and resuspended in 50 μL of transposition mix containing 2.5 μL Nextera Tagment DNA Enzyme (Illumina), 1× Nextera Tagment DNA Buffer (Illumina), and 0.01% digitonin (Promega), using a modified ATAC-seq protocol36 (link). Transposition reactions were performed at 37 °C with 300 r.p.m. mixing for 30 min. Libraries were purified with a QIAQUICK PCR cleanup kit, eluting with 20 μL, and amplified with NEBNext 2 × PCR Master Mix (New England Biolabs) using barcoded PCR primers (Supplementary Table 1 ) for variable numbers of cycles (~10) determined by using qPCR on an aliquot to avoid saturation. Amplified libraries were purified with a QIAQUICK PCR clean up kit and size-selected with a 2.5% agarose gel and MinElute Gel Extraction Kit before high-throughput sequencing on an Illumina HiSeq 2500 (50 bp paired end reads; ELIM Biopharm). Eight technical replicates were performed and data was pooled for peak calling and nucleosome calling (see below).
Nebnext 2 pcr master mix
Manufactured by New England Biolabs
NEBNext 2× PCR Master Mix is a ready-to-use solution for performing high-fidelity PCR amplification. It contains all the necessary components, including DNA polymerase, dNTPs, and reaction buffer, in a single mix.
Automatically generated - may contain errors
Lab products found in correlation
Digitonin, by Promega (2 mentions)
Indexed primers, by New England Biolabs (2 mentions)
Mightyamp dna polymerase, by Takara Bio (2 mentions)
Hiseq 2500 platform, by Illumina (2 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Nextera tagment dna enzyme, by Illumina (1 mentions)
Agencourt ampure xp pcr purification kit, by Beckman Coulter (1 mentions)
Advantage 2 polymerase, by Takara Bio (1 mentions)
Miseq reagent kit v2, by Illumina (1 mentions)
Superscript 3 reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Hiseq 1500 platform, by Illumina (1 mentions)
G9441, by Promega (1 mentions)
Minelute pcr purification kit, by Qiagen (1 mentions)
5 protocols using nebnext 2 pcr master mix
1
ATAC-seq protocol for chromatin accessibility analysis
2
TELP Library Preparation for Single-End Sequencing
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The fC-CET-enriched genomic DNAs were used directly for library preparation using the TELP protocol23 (link). A minor modification was the use of MightyAmp DNA Polymerase (TAKARA) for one round of on-bead primer extension before PCR amplification. The adaptor-ligated samples were then PCR amplified using NEBNext 2× PCR Master Mix (NEB) and indexed primers (NEB). Libraries were checked using the Agilent 2100 Bioanalyzer before loading onto the Illumina Hiseq 2500 platform. A single-end (100 bp or longer) sequencing mode was suggested for maximal data collection.
Two biological replicates of each mESCs were prepared and sequenced, which means that in parallel, two non-enriched input DNAs (Input: preAI), two AI labeled samples (Input: AI) and two pull-down output samples were sequenced simultaneously following the same procedure.
Two biological replicates of each mESCs were prepared and sequenced, which means that in parallel, two non-enriched input DNAs (Input: preAI), two AI labeled samples (Input: AI) and two pull-down output samples were sequenced simultaneously following the same procedure.
3
HLA-DRB Gene Expression Analysis
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RNA isolation of the cell lines Raji, BL64 and KG-1 was performed using the RNeasy Plus Mini Kit (250) from Qiagen according to manufacturer’s instructions. Next, cDNA was synthesized using the Superscript III Reverse Transcriptase Kit from Invitrogen. A 399-bp-long fragment covering exon 2 of HLA-DRB was amplified using the NEBNext 2× PCR Master Mix from New England Biolabs. In a second PCR step, the Illumina flow cell binding site and indices for demultiplexing were added to the amplicons using the Advantage 2 Polymerase from Clontech. Subsequent purification of the PCR products was performed with the Agencourt AMPure XP PCR Purification Kit (Beckman Coulter) according to manufacturer’s instructions. For Illumina Sequencing, the MiSeq V2 Reagent Kit was used with 2 × 250 cycles on a MiSeq machine.
4
TELP Library Preparation for Single-End Sequencing
5
Omni-ATAC-seq Protocol for 93T449 Cells
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ATAC-seq of 93T449 cells was performed according to the Omni-ATAC protocol developed by Corces et al.51 (link). Briefly, 50,000 viable cells were pelleted and lysed in resuspension buffer (Tris-HCl pH 7.4 10 mM, NaCl 10 mM, MgCl2 3 mM, NP40 0.1%, Tween-20 0.1%, and digitonin 0.01%—Promega #G9441) for 3 min on ice. Next, nuclei were pelletted and the transposition reaction was performed incubating the lysate for 30 min at 37 °C under agitation in the presence of transposition mixture (Tris-HCl pH 7.6 10 mM, MgCl2 5 mM, dimethyl formamide 10%, Tn5 enzyme 100 nM—Illumina #20018704, digitonin 0.01%—Promega #G9441, Tween-20 0.1%, and PBS 33%). The transposition reaction was purified (Qiagen MinElute PCR Purification kit, Qiagen #28004) and pre-amplified by PCR in the presence of adapter primers (NEBNext 2× PCR Master Mix, New England Biolabs Inc. #M0541S). Amplification was monitored by RT-PCR using the PowerUp™ SYBR™ Green Master Mix (ThermoFisher Scientific, #A25741) to determine the number of additional amplification cycles89 . Finally, samples were purified with SPRI AMPure XP beads (ThermoFisher Scientific) and sequenced on an Illumina HiSeq 1500 platform in single-end using 50-bp reads.
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