RNA-seq and MNase-seq library preparation and next-generation sequencing
This protocol is extracted from research article:
Cellular response to moderate chromatin architectural defects promotes longevity
Sci Adv, Jul 10, 2019; DOI: 10.1126/sciadv.aav1165

Total RNA was extracted as described above. Poly-A RNA was purified from 5 μg total RNA with Dynabeads Oligo (dT)25 (Thermo Fisher Scientific). Paired-end RNA libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England BioLabs). Sequencing of three biological replicates was performed using the Illumina HiSeq 2500 platform.

Chromatin was digested with 120 U of MNase (New England BioLabs) and 8 U of EXOIII (Promega), as previously described (47), and purified using the QIAGEN Genomic-tip 20/G column. Mononucleosome DNA was separated by agarose gel electrophoresis and purified using the Quantum Prep Freeze ‘N Squeeze DNA Gel Extraction Spin Column (Bio-Rad). Purified DNA was treated with NEBNext End Repair Module, followed by Agencourt AMPure XP beads (Beckman Coulter) cleanup, and dA (deoxyadenosine) tailing with Klenow fragment (New England BioLabs). After purification, the samples were ligated with Illumina TruSeq multiplexing primers using the Quick Ligation Kit (New England BioLabs) and amplified using the KAPA HiFi Ready Mix (Thermo Fisher Scientific). Following the final purification, the DNA was quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). Sequencing was performed on the Illumina HiSeq 2500 platform using three biological replicates.

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