RRBS of human fetal testis treated to a mixture of DEHP/BPA
RRBS of human fetal testis treated to a mixture of DEHP/BPA
At the end of exposure, fetal human testis were collected and each pieces were cleaned from extracellular matrix and murine muscular tissue. Then, testicular pieces were pooled by condition and stored in RLT buffer from AllPrep DNA/RNA microkit (Qiagen).  human fetal testis (from 8-11 WPF) were cut into small pieces (~1 mm3 ) and 4-6 pieces/testis (depending of the age or size of the gonad) were insert into the muscle of the left back of two twin female mice. One week after surgery, mice were orally exposed to chemicals during 6-11 weeks (corresponding to the 18 WPF equivalent age) Xenografted CTL mice (control) were orally exposed (drinking water) to vehicle (ETOH, 1/1000000) and DE/BP mice were exposed to a mixture of DEHP and BPA at 1µM each.  Total DNA from xenografted testes were simultaneously isolated using the AllPrep DNA/RNA microkit (ref : 80284, Qiagen, France) according to manufacturer's instructions. DNA concentrations and quality were assayed with Qbit  fluorometer (Thermofisher) and Agilent bioanalyser. After DNA quantitation, pools of DNA per condition were performed containing from 3 to 8 testis per pool.  RRBS was performed by Integragen SA (Evry, France) with the Diagenode Premium RRBS kit. In brief, 100 ng of qualified genomic DNA were digested with MspI. After end-repair, A-tailing, and ligation to methylated and indexed adapters, the size selected library fragments were subjected to bisulfite conversion and PCR amplified.