GSM1462977: RNA-seq_598-SKD_3dDox_rep3; Homo sapiens; RNA-Seq
For HA-tag ChIP-seq, stable MCF7 cell lines were induced using 100 ng/ml doxycycline (Sigma) at 0 h, doxycycline media was refreshed at 48 h, and cells were harvested at 72 h by crosslinking in a final concentration of 1% formaldehyde. Crosslinking was stopped after 5 min by adding glycine to a final concentration of 125 mM. Crosslinked cells were washed in cold phosphate buffered saline, lysed using 1 ml low-salt IP buffer (150 mM NaCl, 50 mM Tris-HCl (pH7.5), 5 mM EDTA, NP-40 (0.5%), Triton X-100 (1%) containing protease inhibitors) and aliquoted at 1 × 10∧7 cells/ml. For Bisufite-seq, 2 microgram of genomic DNA was isolated using a QIAeasy DNA mini kit (Qiagen, Venlo, The Netherlands) and sonicated using a Covaris to an average molecular weight of 150 bp. Achievement of the desired size range was verified by Bioanalyzer (Agilent) analysis. Fragmented DNA was repaired to generate blunt ends using the END-It kit (Epicentre Biotechnologies, Madison, WI) according to manufacturer’s instructions. Following incubation, the treated DNA was purified using AmpureX beads from Agencourt. In general, magnetic beads were employed for all nucleic acid purifications in the following protocol. Following end repair, A-tailing  was performed using the NEB dA-tailing module according to manufacturer’s instructions (New England Biolabs, Ipswich, MA). Adapters with a 3′ ‘T’ overhang were then ligated to the end-modified DNA. For whole genome bisulfite sequencing, modified Illumina paired-end (PE) adapters were used in which cytosine bases in the adapter are replaced with 5-methylcytosine bases. Depending on the specific application, we utilized either Early Access Methylation Adapter Oligos that do not contain barcodes, or the adapters present in later versions of the Illumina DNA Sample Preparation kits, which contain both indices and methylated cytosines. Ligation was carried out using ultrapure, rapid T4 ligase (Enzymatics, Beverly, MA) according to manufacturer’s instructions. The final product was then purified with magnetic beads to yield an adapter-ligation mix. Prior to bisulfite conversion, bacteriophage lambda DNA that had been through the same library preparation protocol described above to generate adapter-ligation mixes was combined with the genomic sample adapter ligation mix at 0.5% w/w. Adapter-ligation mixes were then bisulfite converted using the Zymo DNA Methylation Gold kit (Zymo Research, Orange, CA) according to the manufacturer’s recommendations. Final modified product was purified by magnetic beads and eluted in a final volume of 20 µl. Amplification of one-half the adapter-ligated library was performed using Kapa HiFi-U Ready Mix for the following protocol: 98º 2′ ; then six cycles of 98º 30′ ′ , 65º 15′′, 72º 60′′; with a final 72º 10′ extension, in a 50 µl total volume reaction. The final library product was examined on the Agilent Bioanalyzer, then quantified using the Kapa Biosystems Library Quantification kit according to manufacturer’s instructions. Optimal concentrations to get the right cluster density were determined empirically but tended to be higher than for non-bisulfite libraries. RNA was harvested using the Qiagen RNeasy kit and Illumina libraries were made with the Illumina TruSeq Stranded mRNA Sample Prep kit according to the manufacturer’s instructions.