cells from preantral follicles maintained for 9 days in vitro
strain
Swiss
gender
female
age
8-weeks
tissue
ovary
cell type
cells from preantral follicles maintained for 9 days in vitro
chip antibody
none
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells grown to confluence in three 150-mm plates were fixed in situ with 1% Formaldehyde for 10 min at room temperature, fixation was then blocked with 125 mM glycine, cells were rinsed and scrapped in PBS. Cells were first lysed in Lysis buffer A (50 mM Hepes pH 7.5, NaCl 140 mM, EDTA 1 mM, glycerol 10% NP-40 0.5% and Triton X-100 0.25%), then spinned and rinsed in lysis buffer B (Tris 10 mM pH 7.5, NaCl 200 mM, EDTA 1 mM, EGTA 0.5 mM), centrifugated again and finally resuspended in lysis buffer C (Tris 10 mM pH 7.5, NaCl 100 mM, EDTA 1 mM, EGTA 0.5 mM, Na–deoxycholate 0.5%, N-lauryl sarcosine 0.5%). Chromatin was sheared in a Bioruptor sonicator, used at high power with 30/30 s cycles for 20 min, then 1% Triton X-100 was added to lysates, and cell debris were centrifugated. In parallel, 30 μg anti-FOXL2 were incubated with 200 μl ProteinG Dynabeads for 4 h in PBS containing 0.1% BSA. Lysates were incubated with the beads overnight, beads were then washed five times in wash buffer (50 mM Hepes pH 7.6, 500 mM LiCl, 1 mM EDTA, 0.7% Na–deoxycholate, 1% NP-40) and once in TBS. DNA–protein complexes were then eluted in 10 mM Tris pH 8, 1 mM EDTA, 1% SDS at 65 °C for 15 min. Crosslinks were reversed at 65 °C overnight, then recovered DNA was treated with RNaseA, proteinase K and purified using phenol–chloroform. Input samples underwent to the same procedure. Adapters were ligated using a SPRI-TE Nucleic Acid Extractor Robot with a SPRIworks Fragment Library Kit I and adapters from TruSeq DNA LT Sample Prep kit (Illumina). Ligated fragments were amplified with C and D primers(C primer :5’-AATGATACGGCGACCACCGAGATCTACAC-3’b and D primer : 5’-CAAGCAGAAGACGGCATACGAGAT-3’) using Phusion HF polymerase (Thermo Fisher Scientific) for 14 PCR cycles. 200 to 400 bp fragments were selected on gel and purified on a Qiagen column.