For RT-qPCR, total RNA was extracted and purified using the Quick-RNA MiniPrep Kit (Zymo Research). The quantity and quality of RNA was determined using a spectrophotometer. RNA was reverse transcribed using the High-Capacity RNA to cDNA Kit (Applied Biosystems) according to the manufacturer’s instructions. For ATAC-qPCR, cells were processed as described above for ATAC-Seq. For qPCR, nuclei were isolated from 50,000 cells and sequencing adapters were transposed for 30 min at 37 °C using 2.5 μl of TDE1 (Nextera Tn5 transposase, Illumina). The reaction was terminated and purified using MinElute reaction cleanup (Qiagen) and used as template. In both cases, qPCR was performed using 1 μl of 5 μM Powerup SYBR green PCR Master Mix (Applied Biosystems), 2 μl of dH2O and 2 μL of cDNA sample in a 10 μl reaction. Each measurement was carried out in a duplicate using a CFX384 Real-Time System (Bio-Rad). The PCR conditions were: 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 60 s. For ATAC-qPCR, primers were designed to flank the center of the DAR as determined by ATAC-Seq. Primer sequences used are listed in Supplementary Table 2 . Gene expression levels were normalized to β-actin, and accessibility changes were normalized to GAPDH.
Minelute reaction cleanup
Manufactured by Qiagen
The MinElute Reaction Cleanup kit is a DNA purification system designed to efficiently remove unwanted reaction components such as salts, primers, and unincorporated nucleotides from enzymatic reactions. The kit utilizes a silica-based membrane technology to selectively bind and recover DNA fragments of 70 base pairs and larger.
Automatically generated - may contain errors
Lab products found in correlation
Quick rna miniprep kit, by Zymo Research (2 mentions)
Cfx384 real time system, by Bio-Rad (2 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (2 mentions)
Nextera tn5 transposase, by Illumina (2 mentions)
Powerup sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Novaseq platform, by Illumina (1 mentions)
Td buffer, by Illumina (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
3 protocols using minelute reaction cleanup
1
Quantitative Analysis of Gene Expression and Chromatin Accessibility
2
ATAC-seq protocol for chromatin accessibility
3
Quantitative gene expression and chromatin accessibility
Check if the same lab product or an alternative is used in the 5 most similar protocols
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For RT-qPCR, total RNA was extracted and purified using the Quick-RNA MiniPrep Kit (Zymo Research).
The quantity and quality of RNA was determined using a spectrophotometer. RNA was reverse transcribed using the High Capacity RNA to cDNA Kit (Applied Biosystems) according to the manufacturer's instructions. For ATAC-qPCR, cells were processed as described above for ATAC-Seq. For qPCR, nuclei were isolated from 50,000 cells and sequencing adapters were transposed for 30 min at 37°C using 2.5 µL of TDE1 (Nextera Tn5 transposase, Illumina). The reaction was terminated and purified using MinElute reaction cleanup (Qiagen) and used as template. In both cases, qPCR was performed using 1 µL of 5 µM
Powerup SYBR green PCR Master Mix (Applied Biosystems), 2 µL of dH2O and 2 µL of cDNA sample in a 10 µL reaction. Each measurement was carried out in a duplicate using a CFX384 Real-Time System (Bio-Rad). The PCR conditions were: 95°C for 2 min, followed by 40 cycles of 95°C for 15 s, and 60°C for 60 s. Primer sequences used are listed in Supplementary Table S2. Gene expression levels were normalized to b-actin, and accessibility changes were normalized to GAPDH.
The quantity and quality of RNA was determined using a spectrophotometer. RNA was reverse transcribed using the High Capacity RNA to cDNA Kit (Applied Biosystems) according to the manufacturer's instructions. For ATAC-qPCR, cells were processed as described above for ATAC-Seq. For qPCR, nuclei were isolated from 50,000 cells and sequencing adapters were transposed for 30 min at 37°C using 2.5 µL of TDE1 (Nextera Tn5 transposase, Illumina). The reaction was terminated and purified using MinElute reaction cleanup (Qiagen) and used as template. In both cases, qPCR was performed using 1 µL of 5 µM
Powerup SYBR green PCR Master Mix (Applied Biosystems), 2 µL of dH2O and 2 µL of cDNA sample in a 10 µL reaction. Each measurement was carried out in a duplicate using a CFX384 Real-Time System (Bio-Rad). The PCR conditions were: 95°C for 2 min, followed by 40 cycles of 95°C for 15 s, and 60°C for 60 s. Primer sequences used are listed in Supplementary Table S2. Gene expression levels were normalized to b-actin, and accessibility changes were normalized to GAPDH.
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