21000 bioanalyzer

Total RNA was isolated using TRIzol (Invitrogen) according to the manufacturer’s instructions. An additional step was introduced to remove calcium (derived from ECM). RNA was precipitated by overnight incubation with 4 M LiCl and 50 mM EDTA at −20°C. After precipitation and centrifugation for 30 min at 14,000 rpm and 4°C, the RNA pellet was washed four times with 70% ethanol and dissolved in H₂O. The RNA concentration was determined spectrophotometrically using a NanoDrop ND-2000 (Thermo Scientific, Wilmington, DE, USA) and its quality accessed by RNA 6000 Nano assay on a 21000 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), both according to the manufacturer’s instructions.

The DNA was extracted using the QIAamp PowerFecal Pro DNA Kit (Qiagen, Crawley, West Sussex, UK) following the manufacturer’s instructions, using 200 ± 50 mg of fecal content from samples. Environmental samples were previously thawed on ice, centrifuged at 15000 rpm for 1 min at 4° C, the supernatant was discarded and pellet was used for DNA extraction. A Qubit fluorometer (Qubit 3, BioSciences, Dublin, Ireland) was used to determine the total DNA concentration. The 2 samples from the different rooms for each type and time point were pooled by adding 5µL of each sample at a concentration of 1ng/µL. Paired-end sequencing libraries were prepared from the extracted DNA using the Illumina Nextera XT Library Preparation Kit (Illumina Inc., San Diego, CA) followed by sequencing on the Illumina NextSeq 500 platform using high-output chemistry (2 × 150 bp) according to the manufacturer’s instructions. Library size from each sample was assessed on an Agilent Technology 21000 Bioanalyzer using a High Sensitivity DNA chip.

PGCs from E13.5 male embryos were sorted by flow cytometry and used for Affymetrix microarray analysis. Three individual samples of 10 000 PGCs (pooled from at least five embryos for each sample) were processed for WT and KO genotypes. PGCs were directly sorted by flow cytometry in lysis buffer from the RNeasy micro kit (Qiagen, Cortaboeuf, France). The quantity and quality of RNA were analyzed using a 21 000 Bioanalyzer (Agilent Technologies, Les Ulis, France). Only samples with an RNA integrity number (RIN) equal to or above 7 were used for the transcriptomic analysis. RNA samples were then analyzed using an Affymetrix GeneChip™ Mouse gene 2.0 ST Array (Thermofisher Scientific, Villebon sur Yvette, France). Our data were then studied with GSEA and Ingenuity Pathway Analysis software. For quantitative RT-PCR, mRNA was prepared using RNeasy® Micro and Mini kits (Qiagen, Cortaboeuf, France). The mRNA was then reverse-transcribed with a Quantitect kit (Qiagen, Cortaboeuf, France). Quantitative RT-PCR was performed using an AB7900 device (Applied Biosystems, Thermofisher Scientific, Villebon sur Yvette, France) with Fast SYBR® Green Master Mix (Applied Biosystems, Thermofisher Scientific, Villebon sur Yvette, France). The primers are listed in Supplementary Material, Table S3.