Labchip gx touch ht

The rhizosphere sediment microbial community DNA was extracted using a classic freeze‐grinding method and purified using a Power Soil DNA Isolation Kit (Mo Bio Laboratories)
⁷³ (link). DNA quality was measured using a Nanodrop (NanoDrop One; Thermo Fisher Scientific), and the absorbance ratios of 260/280 and 260/230 were about 1.8 and above 1.7, respectively. DNA concentrations were quantified using a fluorescent method (Qubit 4 Fluorometer; Thermo Fisher Scientific). Sequencing libraries were prepared using a VAHTSTM Universal DNA Library Prep Kit for Illumina (Vazyme Biotechnology) following the manufacturer's instructions, and the quality was checked using LabChip GX Touch HT (PerkinElmer). Sequencing was performed using an Illumina NextSeq. 550 platform (2 × 150 paired ends) (Illumina). In total, 208,574,599 raw paired‐end (PE) reads (39–49 million reads per sample) were obtained from the KO rhizosphere and 205,091,594 raw PE reads (35–45 million reads per sample) were obtained from the SA rhizosphere (Table S4).

Homozygous mutant males bearing a null mutation of the Tph1 gene (congenic on a C57BL/6 N background) and age-matched wild-type mice were used. The line has been maintained through mating of females heterozygous for the Tph1 gene with heterozygous males obtained from Cyagen Biosciences Inc. Genomic DNA was extracted from tails of littermates using TaKaRa MiniBEST Universal Genomic DNA Extraction kit (Ver.5.0_Code No.9765). Genotypes were confirmed by PCR-LabChip (PerkinElmer LabChip GX Touch HT) analysis using the forward primer F1: 5′-ACATCAGCCTTCTGCTCTGTTTC-3′ and the reverse primer R1: 5′-TCACTGAGAGCATCAAGCCCAG-3′ and R2: 5′-ATTTCCGGGACTCGATGTGTAAC-3′. Tph1 mutant and wild-type alleles correspond to the 611- and 489-bp fragments, respectively.
Before the experiment, animals were all housed (3–5 mice per cage) with free access to water and chow pellets in a light—(12 h on/12 h off; lights off at 2000 h) and temperature—(20–22 °C) controlled environment. The animal studies were conducted in accordance with the institutional guidelines for animal experiments at Tohoku University Graduate School of Medicine and all experimental protocols were approved by the institutional committee at Tohoku University.

Three biological replicates of fresh-frozen sperm samples and cryopreserved sperm samples were used for RNA extraction, which underwent the same thawing procedure (40 °C water bath for 20 s). To achieve high reproducibility, the sperm samples were homogenized using a PowerLyzer24 instrument (Qiagen, Redwood City, CA, USA). Total RNA was extracted using AllPrep DNA/RNA Kit (Qiagen, Redwood City, CA, USA) following the manufacturer’s instructions. RNA concentrations were measured with the NanoDrop OneC Microvolume Spectrophotometer (Thermo Scientific, Waltham, MA, USA). Evaluation of RNA quality was conducted using the LabChip GX Touch HT (PerkinElmer, Hopkinton, MA, USA). RNA-seq library construction was performed following the procedure described in our previous publication [96 (link)] with 500 ng of total RNA input. The size distributions of cDNA libraries were checked using the TapeStation 4200 D1000 ScreenTape (Agilent Technologies, Santa Clara, CA, USA), and library concentrations were determined by a Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). The libraries were commercially sequenced on the Illumina NovaSeq 6000 platform with a 2 × 150 Paired-End configuration at Novogene (Novogene Corporation Inc., Sacramento, CA, USA).

Enriched libraries were quantified using a LabChip GX Touch HT (Perkin Elmer) following the manufacturer’s instructions (Supplementary Note 5), and were pooled in equimolar concentrations in a total volume of 60 μl. To remove low-molecular weight primer dimer and library-build/capture artifacts, fragments between 140 bp and 300 bp were size-selected from the pooled library using two lanes of a Pippin Prep (Sage Science) eGel cassette following the manufacturer’s instructions. The two lanes of size-selected library were recombined, and were purified and concentrated through a QIAGEN PCR Purification kit, following the manufacturer’s instructions, with minor changes (Supplementary Note 5). The final sequencing library was quantified again on the LabChip GX Touch HT. The library was diluted to 4 nM in ultrapure water and was sequenced using Illumina’s high-throughput platform NextSeq, following the manufacturer’s instructions with minor changes (Supplementary Note 5).

High molecular weight (HMW) genomic DNA (gDNA) was extracted from M. zaraptor whole-body samples collected 24 h after eclosion from the AUB colony using Genomic-tip 20/G kit (Qiagen, MD, United States). The DNA concentration was measured on a Qubit 3.0 Fluorometer instrument (Thermo Fisher Scientific, MD, United States). The gDNA quality and the size distribution were assessed on an Agilent TapeStation 4200 machine (Agilent Technologies, CA, USA) with the genomics screentapes. A total of 10 μg high-quality M. zaraptor HMW gDNA was sheared into 20 Kb fragments. After end-repair and ligating the specific adapter oligos, the DNA fragments were annealed with sequencing primer v2 and Sequel II DNA Polymerase, bound to the SMRTbell templates, and the library was prepared using the SMRTbell Template Prep kit v2 with the CCS HiFi Library construction protocol (Pacific Biosciences, CA, United States) at the HudsonAlpha Genome Sequencing Center (HGSC, Huntsville, AL, United States). The concentration and the size distribution for the prepared library were determined on LabChip GX Touch HT (PerkinElmer, MA, United States), and sequenced on a PacBio Sequel II System at HGSC (Supplementary Table S1).