Quantifluor dsdna system

Total RNA was prepared from WT SH and EE brain hemispheres (n = 6 each) using Trifast reagent (Peqlab) according to the protocol of the supplier. As starting material for the library preparation, 0.5 μg of total RNA was used. The libraries were generated according to the TruSeq mRNA Sample Preparation Kits v2 Kit from Illumina (Cat. N°RS-122-2002). The fluorometric based QuantiFluor^TM dsDNA System from Promega (Mannheim, Germany) was used for accurate quantitation of cDNA libraries. The size of final cDNA libraries was determined by using the Fragment Analyzer from Advanced Bioanalytical. cDNA libraries were amplified and sequenced by using the cBot and HiSeq2000 from Illumina (SR; 1 × 50 bp; ca. 30 Mio reads per sample). Sequence images were transformed to bcl files using Illumina software BaseCaller, which were demultiplexed to fastq files with CASAVA v1.8.2 and quality checks were done via fastqc.
Read alignment was performed using STAR v2.3.0 to the hg19 reference genome, while data conversion and sorting was carried out with samtools 0.1.19¹ and reads per gene were counted via htseq version 0.6.1². Data analysis was performed using R/Bioconductor (3.0.2/2.12³) with DESeq2 and gplots packages. Candidate genes were filtered to a minimum of FDR-corrected p-value < 0.05 as described previously (Huttenrauch et al., 2016 (link)).

Library preparation for RNA-Seq was performed using the TruSeq Stranded Total RNA Sample Preparation Kit (Illumina, RS-122-2201) starting from 1,000 ng of total RNA. Accurate quantitation of cDNA libraries was performed by using the QuantiFluor TM dsDNA System (Promega). The size range of final cDNA libraries was determined applying the SS-NGS-Fragment 1–6,000 bp Kit on the Fragment Analyzer from Advanced Analytical (320 bp). cDNA libraries were amplified and sequenced by using the cBot and the HiSeq2000 from Illumina (SR; 50 bp; 35 million reads per sample). Sequence images were transformed with Illumina software BaseCaller to bcl files, which were demultiplexed to fastq files with CASAVA v1.8.2.

As starting material for the library preparation, 0.5 μg of total RNA was used. The libraries were generated according to the TruSeq mRNA Sample Preparation Kits v2 Kit from Illumina (Cat. N°RS − 122-2002). The fluorometric based QuantiFluor™ dsDNA System from Promega (Mannheim, Germany) was used for accurate quantitation of cDNA libraries. The size of final cDNA libraries was determined by using the Fragment Analyzer from Advanced Bioanalytical. cDNA libraries were amplified and sequenced by using the cBot and HiSeq2000 from Illumina (SR; 1 × 50 bp; ca. 30 Mio reads per sample). Sequence images were transformed to bcl files using Illumina software BaseCaller, which were demultiplexed to fastq files with CASAVA v1.8.2 and quality checks were done via fastqc.

For this experiment, 10 wells of barcoding PCR product were pooled and sequenced. Based on mtDNA copy number estimates from mtDNA content per mouse oocyte [25 (link)], this yielded ~100 unique molecules per sample. Products were cleaned using SPRIselect beads (Beckman Coulter Life Sciences, Indianapolis, IN, USA) at 1:4 ratio of product to bead-buffer to diminish the retention of short, non-target by-products. The QuantiFluor dsDNA System (Promega, Madison, WI, USA) was used to quantify DNA concentrations, and 1.5 μg of cleaned DNA was prepared using the 1D amplicon/cDNA by Ligation SQK-LSK-109 protocol (ACDE_9064_v109_revD_23May2018; Oxford Nanopore Technologies, Oxford, United Kingdom) for sequence analysis using a MinION R9 flow cell and MinION software version 19.10.1 (Oxford Nanopore Technologies). Sequence data were base called using Guppy software (version 2.3.7) and the dna_r9.4.1_450bps_flipflop.cfg model (Oxford Nanopore Technologies).

Frozen samples were thawed and centrifuged at 5,500 rpm (6,230 × g) for 10 minutes at 4°C. The pellet in each tube was washed twice with sterile 0.85% saline solution and then resuspended in 1 mL of the saline solution. An aliquot (800 μL) was centrifuged again; the pellet was resuspended in 350 μL of solution 1 from the PowerBiofilm DNA Isolation Kit (Qiagen, Hilden, Germany) and used for the extraction of total DNA in accordance with the manufacturer’s instructions, but with a few modifications. Instead of using beads provided in the kit, 400 μL of 0.5-mm-diameter zirconia beads and two 5-mm-diameter zirconia beads were used to crush bacterial cells. Bacteria were crushed by using Beads Crusher μT-01 (Taitec Corp., Tokyo, Japan) at 4,600 rpm for seven repeats of 60-second run with 60-second intervals. The total DNA was eluted in 100 μL of the elution buffer provided in the kit and stored at –20°C until use. The concentration of DNA was measured with a Quantus Fluorometer (Promega, Madison, WI, USA) with the QuantiFluor dsDNA System (Promega) according to the manufacturer’s instructions. The DNA quality was verified by measuring the ratio of the absorbance at 260 nm and 280 nm (ratio, 1.8–2.0) with a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific).