Instrument: NextSeq 500
Strategy: Bisulfite-Seq
Source: GENOMIC
Selection: RANDOM
Layout: SINGLE
Construction protocol: RNA was extracted using RNeasy mini kit (Qiagen) and RNAse free DNaseI (Sigma) was used to eliminate DNA contamination. For ChIPseq, ESCs were fixed with 1% formaldehyde for 10 minutes. After quenching and cell lysis, chromatin were sonicated using a E220 focused-ultrasonicator (Covaris). Sheared chromatin, 5 µg antibody, and 50 µl protein A/G beads (Santa Cruz) were used for each immunoprecipitation. Immunoprecipitated DNA were purified after washing, eluting, and reverse-crosslinking and submitted for library preparation. For PROseq, nuclei were isolated by Dounce homogenizer with loose pestle. Nuclei were subjected to nuclear run-on in the presence of biotin-11-ATP/GTP/CTP/UTP and Drosophila S2 spike-in nuclei. Biotinylated nascent RNA was purified by Dynabeads M-280 streptavidin. For mRRBS, genomic DNA was digested with MspI (New England BioLabs) before size selection of 100- to 250-bp fragments with solid-phase reversible immobilization beads (MagBio Genomics). DNA was bisulfite converted with the EZ DNA Methylation-Lightning Kit. ChIP-seq libraries were prepared using the KAPA HTP Library Preparation Kit complemented with NEXTflex DNA Barcodes from Bioo Scientific. 10 ng of DNA was used as starting material for input and ip samples. Libraries were amplified using 13 cycles on the thermocycler. Post amplification libraries were size selected at 250- 450bp in length using Agencourt AMPure XP beads from Beckman Coulter. Libraries were validated using the Agilent High Sensitivity DNA Kit. RNA-seq libraries were prepared using the Illumina TruSeq Stranded Total RNA Preparation Kit with Ribo-Depletion. Input RNA quality was validated using the Agilent RNA 6000 Nano Kit. 200ng-1 µg of total RNA was used as starting material. Libraries were validated using the Agilent DNA 1000 Kit ChIP-seq libraries were prepared using the KAPA HTP Library Preparation Kit complemented with NEXTflex DNA Barcodes from Bioo Scientific. 10 ng of DNA was used as starting material for input and ip samples. Libraries were amplified using 13 cycles on the thermocycler. Post amplification libraries were size selected at 250- 450bp in length using Agencourt AMPure XP beads from Beckman Coulter. Libraries were validated using the Agilent High Sensitivity DNA Kit. RNA-seq libraries were prepared using the Illumina TruSeq Stranded Total RNA Preparation Kit with Ribo-Depletion. Input RNA quality was validated using the Agilent RNA 6000 Nano Kit. 200ng-1 µg of total RNA was used as starting material. Libraries were validated using the Agilent DNA 1000 Kit