Q bit fluorometer

Total RNA was isolated from testicular tissue using the miRCURY™ RNA Isolation Kit (Cat No. 300111, Exiqon Inc. Vedbaek, Denmark). It is based on spin column chromatography using a proprietary resin as the separation matrix. This method of purification of total RNA allows for the isolation of the very small (<200 nucleotide) RNA fraction.
In brief, the frozen tissue is completely homogenized in a lysis solution using TissueRuptor. The lysate is incubated with Proteinase K for protein removal and loaded on a spin column. The removal of genomic DNA is performed directly on the column with DNase I (Qiagen, Venlo, Limburg) at a final concentration of 0.25 Kunitz unit/μL. Eluted total RNA was either used for cDNA synthesis or stored in – 80° Celsius. RNA concentration was measured with a fluorescence based quantitation assay using Qbit® fluorometer (Life Technology™, NY, USA), and the integrity of RNA was evaluated by calculating RNA integrity number (RIN, based on the detection of 18S and 28S and the amount of degradation products) on Agilent Bioanalyzer 2100 (Aligent Technologies, CA, USA). Only RNAs with RIN greater to or equal to 7 were used in the expression studies, this indicate that the RNA sample has minimal degradation products.

Tissue depleted of DA neuronal regions after LCM was also collected from the slides in 100 µl of lysis/denaturing buffer from the RNAqueous micro kit (Life Technologies, Zug, Switzerland). RNAs were extracted following the manufacturer protocol, quantified with a Qbit™ fluorometer (Thermo Fisher, Waltham, MA, US), and analyzed with an Agilent 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA) to check the RNA profile and obtain the RNA integrity number (RIN).

Genomic DNA from PHSats was isolated as described above and target site amplification was carried out using Q5 High Fidelity DNA Polymerase (New England Biolabs) and primers as indicated in Table S6. PCR products were run on an agarose gel und gel purified using NucleoSpin Gel and a PCR Clean-up kit (Machery-Nagel). DNA concentration was measured using a Qbit fluorometer (Thermo Fisher Scientific) and the concentration was adjusted to 20 ng/μL. Next-generation sequencing (Amplicon EZ-service) was performed with GENEWIZ (Leipzig, Germany) using an Illumina MiSeq platform with 2 × 250 bp paired-end reads. Sequencing results were analyzed using CRISPResso2.⁵⁸ (link) The following parameters were applied: minimum homology for alignment to an amplicon: 60%; center of the quantification window (relative to the 3′ end of the provided sgRNA): −3; quantification window size (bp): 1; minimum average read quality (phred33 scale): >30; minimum single bp quality (phred33 scale): no filter; replace bases with N that have a quality lower than (phred333scale): no filter; exclude bp from the left side of the amplicon sequence for the quantification of the mutations: 15 bp; exclude bp from the right side of the amplicon sequence for the quantification of the mutations: 15 bp.

Indexed DNA library for NGS was prepared using the TruSeq® Nano DNA HT Library Preparation Kit (Illumina, cat. 20,015,965) according to manufacturer’s recommendation. Indexed libraries were then quantified using a Q-bit fluorometer (Thermo Fisher Scientific) and pooled together at an equal concentration. The pooled library was diluted to 18 pM concentration. Sequencing was performed on an Illumina MiSeq® using 2 × 300 PE chemistry according to manufacturer’s protocol. The reads were de-multiplexed with the MiSeq Reporter software and were stored as FASTQ files for downstream analysis (Additional file 14: Table S7).