Four SMRT libraries were generated from collected tissues, each corresponding to a specific stage of testis or AG development: T_S2 (testis at the spermatid stage), T_S3 (testis at the sperm stage), AG_S2 (AG at the synthesis stage), and AG_S3 (AG at the secretion stage). The libraries were constructed using the SMRTbell Template Prep Kit from Pacbio (United States). The mRNA was first converted to cDNA using the SMARTer PCR cDNA Synthesis Kit from Takara (United States). The cDNA was then amplified by PCR and purified using AMPure PB beads from Pacbio (United States). Finally, the resulting templates were sequenced on a Sequel II instrument from Pacbio (United States).
Sequel 2 instrument
Manufactured by Pacific Biosciences
Sourced in United States, Sweden
The Sequel II instrument is a DNA sequencing platform developed by Pacific Biosciences. It is designed to generate high-quality long reads for various genomic applications. The Sequel II instrument utilizes Single Molecule, Real-Time (SMRT) sequencing technology to provide accurate and efficient DNA sequencing capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Ampure pb bead, by Pacific Biosciences (1 mentions)
Smarter pcr cdna synthesis kit, by Takara Bio (1 mentions)
Smrtbell template prep kit, by Pacific Biosciences (1 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (1 mentions)
Short read sequencing, by Illumina (1 mentions)
Smrtbell express template prep kit 2, by Pacific Biosciences (1 mentions)
Sageelf, by Sage Science (1 mentions)
Qubit 2, by Thermo Fisher Scientific (1 mentions)
La taq polymerase, by Takara Bio (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Rnaeasy mini qiacube kit, by Qiagen (1 mentions)
Iso seq express template preparation protocol, by Pacific Biosciences (1 mentions)
Genomic dna extraction kit, by Qiagen (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Genomic tip columns 500 g, by Qiagen (1 mentions)
Nanodrop nd 1000 spectrometer, by Avantor (1 mentions)
Femto pulse system, by Agilent Technologies (1 mentions)
5 protocols using sequel 2 instrument
1
Single-Molecule Sequencing of Testis and Accessory Gland Development
2
Optimal Representation of Sheep Breed Diversity
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Samples were selected to form an optimal representation of the diversity of European and Asian sheep breeds. The sampling information is provided in Supplemental Tables S2 and S17 . High-quality genomic DNA (gDNA) was extracted from fresh blood samples from 15 individual sheep as previously described (Li et al. 2021 (link)) and assessed for purity and quantity using NanoDrop 1000 (Thermo Fisher Scientific) and Qubit (Thermo Fisher Scientific) assays. Libraries with an average insert size of ∼15 kb were generated using the SMRTbell Express Template Prep Kit 2.0 (PacBio) and fractionated on the SageELF (Sage Science) into narrow library fractions. Libraries were then sequenced on 2-3 SMRT Cells 8M on a Sequel II instrument (PacBio) using 30-h movie times at Annoroad Gene Technology Co., Ltd. Raw data were processed using the CCS algorithm (version 6.0.0, parameters: ‐‐minPasses 3 ‐‐minPredictedAccuracy 0.99 ‐‐maxLength 21,000) to generate highly accurate HiFi reads.
The 15 sheep were additionally sequenced to an average depth of 24× using Illumina short-read sequencing, which was used to estimate the base accuracy of de novo assemblies.
The 15 sheep were additionally sequenced to an average depth of 24× using Illumina short-read sequencing, which was used to estimate the base accuracy of de novo assemblies.
3
Amplification and Sequencing of Eukaryotic rDNA
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We amplified a ~4,500 bp fragment of the rDNA operon using the general eukaryotic primers 3NDf85 (link) and 21R86 (link), including part of the 18S gene, the complete internal transcribed spacer (ITS) region, and part of the 28S gene85 (link),86 (link). PCRs were performed with sample-specific tagged-primers using the Takara LA Taq polymerase (Takara) and 5 ng of DNA as input. PCR-cycling conditions included an initial denaturation step at 94 °C for 5 min, at least 25 cycles of denaturation at 98 °C for 10 s, primer annealing at 60 °C for 30 s and elongation at 68 °C for 5 min and finishing with a final elongation step at 68 °C for 10 min. We limited the number of PCR cycles to 25, where possible, to reduce chimaera formation87 (link). For samples that did not get amplified, we increased the number of cycles to 30. PCR products were assessed using agarose gels and Qubit 2.0 (Life Technologies) and then purified with Ampure XP beads (Beckman Coulter). Amplicons from replicates, size fractions and different sites from the same sampling location were pooled at this stage. SMRTbell libraries were constructed using the HiFi SMRTbell Express Template Prep Kit 2.0. Long-read sequencing was carried out at SciLifeLab (Uppsala, Sweden) on the Sequel II instrument (Pacific Biosciences) on a SMRT Cell 8 M Tray (v.3), generating four 30-h movies.
4
Iso-Seq Express Template Preparation Protocol
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Total RNA was isolated from corneal endothelial tissue using the RNAeasy Mini QIAcube Kit (Qiagen, Valencia, CA). Samples were prepared using the library preparation protocol for sequencing as described in the Iso-Seq Express Template Preparation protocol (Pacific Biosciences, Menlo Park, CA) found in the Procedure & Checklist–Iso-Seq Express Template Preparation for Sequel and Sequel II Systems (Pacific Biosciences, Menlo Park, CA). This protocol can be found at “https://www.pacb.com/wp-content/uploads/Procedure-Checklist-Iso-Seq-Express-Template-Preparation-for-Sequel-and-Sequel-II-Systems.pdf .”. Each sample was sequenced in a single SMRTCell on the Pacific Biosciences Sequel II instrument, using 30 hour movies. Corresponding data can be found in the National Center for Biotechnology Information (NCBI) BioSample database under accession numbers SAMN21432327, SAMN21432328, SAMN21432329, SAMN21432330, SAMN21432331, SAMN21432332.
5
Genomic DNA Extraction from P. giblindavisi
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Nematodes of the inbred P. giblindavisi strain RS5555 were grown on nematode growth medium. Worms were washed off of 100 fully grown plates using M9 buffer and were gently pelleted by centrifugation at 1,300× g for 1 min. The pellet was washed twice in M9 before worms were frozen in liquid nitrogen and ground to a fine powder using a mortar and pestle. The powder was directly transferred into the lysis buffer from the Qiagen genomic DNA extraction kit, which was used in combination with Qiagen genomic tip columns (500/G) (Qiagen, Hamburg, Germany). The protocol was performed following the manufacturer’s instructions. All steps involving sample vortexing were replaced by sample inverting to limit unwanted DNA shearing. DNA quality and quantity were determined with a NanoDrop ND 1000 spectrometer (PeqLab, Erlangen, Germany), a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, USA), and by a Femto pulse system (Agilent, CA, USA). A total of 15 μg genomic DNA was sheared to a target fragment size of 13 kb using a Megaruptor 2 device (Diagenode, Denville, USA). A 13-kb template library was prepared using the BluePippin size-selection system according to the manufacturer's protocol (P/N 100-286-000-07, Pacific Biosciences, CA, USA). The final library was sequenced on half of an SMRT cell of a Pacific Biosciences Sequel II instrument following the Magbead loading protocol.
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