Input samples were 400K primary cortical neurons per biological replicate. Neurons were collected by scraping in cold lysis buffer (10 mM Tris-Cl (pH 7.5), 10 mM NaCl, 3 mM MgCl2, 0.1% (vol/vol) NP-40, 0.1% (vol/vol) Tween-20 and 0.01% (vol/vol) digitonin) and washed in wash buffer (10 mM Tris-Cl (pH 7.5), 10 mM NaCl, 3 mM MgCl2 and 0.1% (vol/vol) Tween-20). Transposition was performed with Tagment DNA TDE1 (Illumina, 15027865). Transposition reactions were cleaned with AMPure XP beads (Beckman, A63880), and libraries were generated by PCR with NEBNext High-Fidelity 2× PCR Master Mix (NEB, M0541). Prior to sequencing, library size distribution was confirmed by capillary electrophoresis using an Agilent 4200 TapeStation with high sensitivity D1000 reagents (5067–5585), and libraries were quantified by qPCR using a KAPA Library Quantification Kit (Roche 07960140001). Libraries were sequenced on an Illumina NextSeq550 instrument (42-bp read length, paired end).
Nebnext high fidelity 2 pcr master mix
Manufactured by New England Biolabs
Sourced in United States
The NEBNext High-Fidelity 2× PCR Master Mix is a ready-to-use solution for high-fidelity PCR amplification. It contains a proofreading DNA polymerase and optimized buffer components to provide efficient and accurate DNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Ampure xp bead, by Beckman Coulter (16 mentions)
Minelute pcr purification kit, by Qiagen (14 mentions)
Minelute kit, by Qiagen (7 mentions)
Novaseq 6000, by Illumina (6 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (6 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (5 mentions)
Nextseq 550, by Illumina (4 mentions)
Hiseq 4000, by Illumina (4 mentions)
Tn5 transposase, by Illumina (4 mentions)
Qiaquick pcr purification kit, by Qiagen (4 mentions)
Nextera dna library prep kit, by Illumina (4 mentions)
Td buffer, by Illumina (4 mentions)
Tagment dna buffer, by Illumina (3 mentions)
Agencourt ampure xp bead, by Beckman Coulter (3 mentions)
Minelute pcr kit, by Qiagen (3 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (3 mentions)
Bioanalyzer 2100 system, by Agilent Technologies (3 mentions)
Spriselect reagent kit, by Beckman Coulter (3 mentions)
Tagment dna enzyme, by Illumina (3 mentions)
Hiseq 3000 system, by Illumina (3 mentions)
70 protocols using nebnext high fidelity 2 pcr master mix
1
Single-cell ATAC-seq protocol for primary neurons
2
Template-Switching Reverse Transcription
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A template-switching reverse transcription reaction (10-μl total volume) contained 0.1 μm RNA template (5′-OH, 5′-p, or 5′-m7G–capped 4rN-25mer-RNA-FAM), 1× Template Switching RT Buffer, 1 mm dNTP solution mixture, 30 nm i7 primer, 1 μm biotin-i5 rGrGrG-3′ TSO (see supporting information for TSO sequence), and 100 units of Template Switching RT. The reaction was performed at 42 °C for 90 min followed by a 10-min heat-denaturation step at 72 °C. Then 1 μl of the reverse transcription reaction was subjected to PCR amplification with NEBNext Universal and Index primers (catalog number E7335) using NEBNext High-Fidelity 2× PCR Master Mix (catalog number M0541). The libraries were cleaned up with NEBNext Sample Purification Beads (catalog number E7767) and sequenced on an Illumina iSeq-100 as described above.
3
ATAC-seq Protocol for Chromatin Accessibility
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ATAC-seq analyses were performed according to the published protocol68 (link). Briefly, 50,000 cells were collected, and nuclei were prepared. The ATAC reactions with Transposase (Illumina) were performed at 37 °C for 30 min. The transposed fragments were purified with a DNA Clean & Concentrator-5 Kit (ZYMO RESEARCH), amplified by PCR using NEBNext High-Fidelity 2× PCR Master Mix (NEB) with sequence primers, and sequenced by Mi-seq (Illumina). The sequenced data were aligned using the Bowtie2 program (v. 2.3.4.3)69 (link) to the human genome (build hg19, GRCh37), and PCR duplicates were removed using MarkDuplicates (Picard v. 1.136 http://broadinstitute.github.io/picard/ ). We made TDF files normalized with read/million, using the Integrative Genomics Viewer (IGV v. 2.3.68)61 (link),62 (link) tools with parameters set to count -z 5 -w 25 -e 150. The peak signals were visualized using IGV. Pearson’s correlation coefficients were calculated by the BamCompare module of the deeptools program70 (link), with a bin size of 150 bp.
4
RNA-seq Library Preparation from cDNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
First-strand cDNA was synthesized as described previously in reverse transcription. Starting from second-strand cDNA synthesized, the RNA-seq library prep kit (VAHTS NR604) was used under the manufacturer's instructions. Twenty-five microliters of first-strand cDNA was mixed with 25 μL second-strand synthesis buffer with dUTP and 15 μL second-strand synthesis enzyme super mix. Samples were incubated for 30 min at 16°C followed by 15 min at 65°C. Adaptors were then ligated to dsDNA by using 25 μL rapid ligation buffer, 5 μL rapid DNA ligase 2, and 5 μL 10 mM adaptors for 15 min at 20°C. Adaptors were then removed by a sequential 0.45× and 1× VAHTS DNA clean beads purification. Samples were dissolved in 19 μL H2O and mixed with 5 μL PCR primer mix, 25 μL NEBNext high-fidelity 2× PCR master mix (NEB M0541S), 1 μL heat-labile UDG. Samples were amplified for 10 min at 37°C; for 30 sec at 98°C; 10 cycles of 10 sec at 98°C, 30 sec at 60°C, and 30 sec at 72°C; followed by 5 min at 72°C. PCR product was purified by 0.9× VAHTS DNA clean beads before sending to sequencing.
5
ATAC-seq Library Preparation from Microglia
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ATAC-seq libraries were prepared as previously described18 (link),23 (link),52 (link),53 (link) with approximately 50,000 sorted microglia. Cells were lysed in 150 µl lysis buffer (10 mM Tris–HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2 and 0.1% IGEPAL CA-630 in water). Resulting nuclei were centrifuged at 500g for 10 min. Pelleted nuclei were resuspended in 50 μl transposase reaction mix (1× Tagment DNA Buffer (Illumina 15027866) and 2.5 μl DNA enzyme I (Illumina 15027865)) and incubated at 37 °C for 30 min. DNA was purified with Zymo ChIP DNA concentrator columns (Zymo Research D5205), eludated with 11 µl of elution buffer, and amplified using NebNext High-Fidelity 2× PCR Master Mix (New England BioLabs M0541) with the Nextera primer Ad1 (1.25 µM) and a unique Ad2.n barcoding primer (1.25 µM) for 8–12 cycles. Resulting libraries were size selected by gel excision to 155–250 bp, purified and single end sequenced using a HiSeq 4000 (Illumina) for 51 cycles according to the manufacturer’s instructions.
6
Targeted Indel Analysis with Illumina Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Targeted indel analysis was performed by amplifying genomic regions of interest with NEBNext High-Fidelity 2× PCR Master Mix (NEB) using a two-round PCR strategy to add Illumina P5 adaptors and unique sample-specific barcodes. Libraries were sequenced with 1 × 200-cycle MiSeq runs (Illumina). Indel rates were measured using Outknocker 224 (link).
(http://www.outknocker.org/outknocker2.htm ).
(
7
ATAC-seq Protocol for Chromatin Accessibility
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ATAC-seq was performed from two biological replicates at four time points as described (Buenrostro et al., 2013 (link)). Briefly, 5 × 104 cells at each time point were harvested and treated with Nextera Tn5 Transposase (Illumina, FC-121–1030) for 45 min at 37°C. Library fragments were amplified using 1× NEBNext High-Fidelity 2× PCR Master Mix (NEB, M0541S) and 1.25 μM of custom Nextera PCR primers 1 and 2. PCR amplification was done with 11 cycles, determined by KAPA Real-Time Library Amplication Kit (Peqlab, KK2701) to stop prior to saturation. Then, the samples were purified using Qiagen MinElute PCR Purification Kit (Qiagen, 28004) and with Agencourt AMPure XP beads (Beckman Coulter, A63881) in 3:1 ratio. The libraries were sequenced at 2 × 50 bp on HiSeq2000 (TAL, Göttingen). The average read numbers obtained were 3 × 107.
8
ATAC-seq analysis of LSKs treated with OF-1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LSKs (10000–20000 cells) were treated with vehicle (DMSO) or OF-1 (10 μM) for 7 days and then sorted by FACS. Library amplification was performed with the NEBnext High Fidelity 2× PCR Master Mix (M0541S, New England Biolabs). Prepared ATAC-seq libraries were sequenced with single-end 50-bp reads on the HiSeq 2000 platform (Suganuma et al., 2016 (link)). Raw reads were adaptor-trimmed and aligned to the genome. Peaks were called using the MACS2 software (v2.1.0.20140616) with default parameters.
9
Omni-ATAC-seq Protocol for C3H/10T1/2 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The samples are prepared according to the methods previous described [75 (link)–77 (link)] with minor modifications. 50,000 C3H/10T1/2 cells were collected for Omni-ATAC-seq. Amplification was performed with NEBNext High-Fidelity 2× PCR Master Mix (NEB, M0541s) and Nextera PCR Primers (8 cycles). The sequences of PCR primers with index adapters are listed in the Supplementary Table 31 . The library pool was sequenced by Rockefeller University genomic core using a NextSeq High Output platform with 75bp paired-end reads in duplicates. An average of 30–40 million paired reads was generated per sample.
10
ATAC-seq Library Preparation Protocol
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!