Nebnext chip seq kit

ChIPseq from G2 and M cells was performed using DNA recovered from pS2 IP and ~ 10 ng of sonicated INPUTs as starting material (see below for ChIP protocol details). Libraries were prepared using the NEBNext ChIP-Seq kit (NEB6240S) in combination with the NEBNext Multiplex Oligo Set (NEB7335L) following the manual specifications. Amplified libraries were submitted for QC and sequencing to Genewiz.
EU-RNA from G2, metaphase and G1 cells treated or not with Auxin and doxycycline were analyzed in duplicated (Auxin vs Mock). For library construct we used EU-labelled RNA purified from ribo-depleted samples as described above and the NEBNext Ultra Directional RNA Library Prep Kit (NEB) following the manual specifications. Amplified libraries sizes were evaluated by loading into an 8% native PAGE and gel bands excised and purified overnight in a NH₄Ac/SDS (0.45 M/0.045%) solution. Libraries were finally precipitated with isopropanol and Glycogen (Thermofisher, 10814010), dissolved in nuclease-free water and submitted to the MGH-sequencing facility for QC and Illumina sequencing.

Total RNA isolation and RNA sequencing were performed as previously described (113 (link)): RNA was extracted using the ARCTURUS PicoPure RNA Isolation Kit (Life Technologies) including a DNase treatment step (Qiagen) following manufacturer’s instructions. cDNA was generated with 1 ng of total RNA and thirteen cycles of amplification using the SMARTer Ultra Low RNA Kit for Illumina sequencing (Clontech) according to manufacturer’s instructions. The sequencing libraries were generated using the NEB Next ChIP-Seq kit (New England Biolabs) according the manufacture’s instructions. The quality of total RNA, cDNA and library were monitored throughout the process using a 2100 Bioanalyzer (Agilent). Sequencing was done in a HiSeq2000 sequencer (Illumina) with three samples per lane, paired-end 100bp.