RNAseq of human fetal testis treated to a mixture of DEHP/BPA
RNAseq 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 RNeasy minikit (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 and RNA from xenografted testes were simultaneously isolated using the AllPrep DNA/RNA microkit (ref : 80284, Qiagen, France) according to manufacturer's instructions. DNA and RNA concentrations and quality were assayed with Qbit  fluorometer (Thermofisher) and Agilent bioanalyser. After DNA and RNA quantitation, pools of DNA or RNA per condition were performed containing from 3 to 8 testis per pool.  RNA sequencing was carried out in triplicate under condition (CTL and DE/BP). RNA-Seq libraries are prepared  by Integragen SA (Evry, France) with NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina according to supplier recommendations (NEB). The capture is then performed on cDNA libraries with the Twist Human Core Exome +Custom IntegraGen Enrichment System according to supplier recommendations (Twist Bioscience). Then a capture of the transcriptome coding regions is performed and the resulting library is suitable for subsequent cluster generation and sequencing. Briefly, the RNA is fragmented into small pieces using divalent cations under elevated temperatures. CDNA is generated from the cleaved RNA fragments with random priming during first and second strand synthesis and sequencing adapters are ligated to the resulting double-stranded cDNA fragments and enriched by 7 PCR cycles. The transcriptome coding regions are then captured from this library using sequence-specific probes to create the final library. For that purpose 500 ng of purified libraries are hybridized to the Twist oligo probe capture library for 16 hr in a singleplex reaction. After hybridization, washing, and elution, the eluted fraction is PCR-amplified with 8 cycles, purified and quantified by QPCR to obtain sufficient DNA templates for downstream applications.