alias: GSM2050798
title: GSM2050798: S2_tandem_HS_R2; Drosophila melanogaster; Bisulfite-Seq
library_construction_protocol: Footprinting protocol was adapted from (Bell et al. 2010; Kelly et al. 2012). 2.5 10^6 Drosophila cells (S2 or OSC) were resuspended in ice-cold lysis buffer (10mM Tris, 10mM NaCl, 3mM Mgcl2, 0.1mM EDTA, 0.5% NP40), incubated 10 min on ice, spun down. Nuclei were washed (10mM Tris, 10mM NaCl, 3mM Mgcl2, 0.1mM EDTA) and resuspended in McviPI reaction buffer (NEB). The nuclei were then incubated with 200U of McviPI (NEB-M0227L) at 30°C for 7.5 min (in presence of 0.6mM SAM, and 300mM Sucrose). The reaction was resuplemented with 100U of McviPI and 128 pmol of SAM before a second incubation round of 30°C for 7.5 min. 10mM MgCl2, 128pmol of SAM and 60U of SssI were added for a third incubation round of 30°C for 7.5 min. Reaction was stopped by adding a SDS-containing buffer (20mM Tris, 600mM NaCl, 1%SDS 10mM EDTA), DNA was extracted after overnight Proteinase K (200 µg/ml) digestion at 55°C. For single treatments, only one of the enzymes was used in the same conditions. Whole-genome bisulfite libraries were prepared using Illumina TruSeq DNA sample preparation kit following manufacturer recommendation. 5g of sheared DNA (Covaris) was used as an input for end-repair and A-tailing. Methylated adaptors were ligated and large DNA fragments (400-600bp) were selected on a low melting agarose gel (BIO-RAD -161-3107). The extracted material was used as an input for bisulfite conversion (Qiagen - 59104). The converted DNA was amplified by 10 cycles of PCR using Pfu Cx HotStart (Agilent - 600410) using Illumina primers. PCR product was purified using AMPureXP beads (Beckman Coulter - A63880) and controlled on Bioanalyser High sensitivity (Agilent 5067-4626). The samples were run on an Illumina HiSeq 2500 generating 150bp paired-end reads. Amplicon bisulfite sequencing was performed as previously described (Baubec et al. 2015). Bisulfite amplicons libraries were prepared using NEBNext ChIP kit and sequenced on a MiSeq instrument generating 250bp paired-end reads.
identifiers.namespace: GEO
identifiers.id: GSM2050798