title: GSM2593606: rpb1-Y-A,FSP+KKR+, Input DNA [13764X6]; Saccharomyces cerevisiae; [Candida] glabrata; ChIP-Seq
library_construction_protocol: Cells were subjected to cross-linking with formaldehyde (1%) for 20 min at room temperature. Glycine (0.125 mM) was added to quench crosslinking and cells were washed with 1X phosphate-buffered saline. For the reference genome, Candida glabrata (ATCC® 36909) was grown at 25°C and cross-linked with formaldehyde as described above. Cross-linked S. cerevisiae (40 × 10^7) and C. glabrata (20 × 10^7) cells were mixed in FA140+SDS buffer (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 140 mM NaCl, 0.1% SDS) and lysed by bead beating. Lysates from two sets of S. cerevisiae and C. glabrata mixture (120 × 10^7 cells) were pooled for sonication using a Diagenode Bioruptor™ (6 cycles of 5 min each, 30 sec ON and 30 sec OFF at high setting). Soluble chromatin was obtained by two sequential high-speed centrifugations. To remove SDS, soluble chromatin obtained from four batches of yeast cells were pooled and transferred to a 5 mL Float-A-Lyzer G2 dialysis device (3.5 - 5 kD MWCO) (Spectra/Por), dialyzed initially for 2 h in 1 L FA buffer (50 mM HEPES-KOH, pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate) with 140 mM NaCl followed by an overnight dialysis against 4 L of the same buffer. The dialysate was centrifuged at 13,000 rpm for 15 min at 4°C. A 100 ml aliquot was set aside for isolating input DNA. The remaining dialysate was split equally into three tubes and pre-cleared using 50 ml of blocked Dynabeads® Protein A for 1h at 4°C with end-over-end rotation. Blocking of Dynabeads® Protein A beads was performed at 4°C with end-over-end rotation for 4h using FA buffer containing 140 mM NaCl, bovine serum albumin (BSA) (0.2 mg/mL, New England Biolabs) and fish skin gelatin (0.5%, Sigma). Pre-cleared dialysate was combined with the custom-made rabbit polyclonal a-Spt6 antibody (30 ml; McCullough 2015). Cross-reactivity of the antibody, which was raised against S. cerevisiae Spt6, for C. glabrata Spt6 was verified by western blot (Figure S6). For a background control, the additional set of pre-cleared dialysate prepared from the wild type control strain was combined with pre-immune serum (30 ml). 100 ml blocked Dynabeads® Protein A was then added and incubated overnight at 4°C with end-over-end rotation. Beads were sequentially washed 5 min each with end-over-end rotation at 4°C with FA buffer + 140 mM NaCl (three washes), FA buffer + 1000 mM NaCl (two washes), FA buffer + 500 mM NaCl (two washes), and one wash each with LiCl/NP40 buffer (10 mM Tris.Cl, pH 8.0, 250 mM LiCl, 0.5% NP40, 0.5% sodium deoxycholate) and TE (10 mM Tris.Cl, pH 8.0, 1 mM EDTA). Elution and reverse crosslinking of ChIP and input DNA followed by RNase A and Proteinase K digestion steps were performed as described previously (Chandrasekharan et al., 2011). ChIP and input DNA were purified using the Qiagen MinElute PCR Purification kit according to the manufacturer's instructions, except two washes each with buffer QG and PE were performed. Sequencing libraries were constructed using NEBNext® Multiplex Oligos (Index Primers Set 1 and Set 2) and NEBNext® ChIP-seq Library Prep Reagent Set for Illumina, and subjected to 50 bp single-end sequencing in an Illumina Hiseq2000 sequencer. HiSeq 50 Cycle Single-Read Sequencing version 4: Sequencing libraries (25 pM) were chemically denatured and applied to an Illumina HiSeq v4 single read flow cell using an Illumina cBot.  Hybridized molecules were clonally amplified and annealed to sequencing primers with reagents from an Illumina HiSeq SR Cluster Kit v4-cBot (GD-401-4001).  Following transfer of the flowcell to an Illumina HiSeq 2500 instrument (HCSv2.2.38 and RTA v1.18.61), a 50 cycle single-read sequence run was performed using HiSeq SBS Kit v4 sequencing reagents (FC-401-4002)
attributes.tag: GEO Accession
attributes.value: GSM2593606