GSM3073971: PolII_522_S2_37_rep2; Drosophila melanogaster; ChIP-Seq
Total RNA was extracted using Trizol and QIAGEN RNeasy kit. DNA after ChIP protocol was isolated using QIAGEN PCR purification kit. ChIP-sequencing 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-sequencing  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 PRO-seq was performed according to the previously published protocol (Mahat et al., 2016a) with minor modifications. All 4 biotinylated nucleotides were used at 25 μM each final concentration for the run-on reaction. RPPH (NEB) was used to remove the 5' RNA cap. Libraries were size selected using a 2% agarose gel on a Pippin HT programmed to elute 140–350 bp. 4sU-seq Cell Labeling with 4sU, RNA Extraction and Fragmentation 20-50 million cells were treated with flavopiridol for 1 hour and washed with PBS twice. The cells were labeled in media with 500 μM 4-thiouridine (4sU, Sigma-Aldrich, St. Louis, MO, USA.) and harvested through centrifugation for 3 min at 3,000 rpm. RNA was extracted with 4 mL Trizol (Invitrogen) and 5 μL 20 mg/mL glycogen. The extracted RNA was further fragmented by base hydrolysis in 0.2 M NaOH on ice for 18 min, neutralized by adding 1x volume of 1 M Tris-HCl pH 6.8 and precipitated with isopropanol.
GEO Accession
GSM3073971