B. sauveli samples of bone and teeth were digested in a EDTA, urea, proteinase K based buffer as described in Ersmark et al. (2015) . B. sauveli samples of horn and dried soft tissue were digested in a DTT, proteinase K based buffer as described in Gilbert et al. (2007) (link). These individual digests of bone, teeth, horn and dried soft tissue were purified as described in Dabney et al. (2013) (link), however using a modified binding buffer as in Allentoft et al. (2015) (link). DNA from a B. javanicus sample of hair shafts were extracted using the DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany) following the manufacturer’s protocol. Purified DNA extracts of B. sauveli were incorporated into libraries following the single tube protocol (Carøe et al., 2017 ) with the reaction setup modifications of Mak et al. (2017) . Specimen B._sauveli_NHMD_231145 was sequenced on one lane of BGISEQ-500 - SR100, as well as 55% of a lane of Illumina HighSeq 2500 - SR80. Specimen B._sauveli_NHMD_231146 was sequenced on two lanes of BGISEQ-500 - SR100. The B. javanicus DNA extract was incorporated into a sequencing library following (Meyer and Kircher, 2010 ) and sequenced on one lane of Illumina HighSeq 2500 - SR100. Details on sequencing output is given in Table S1 , sheet Mapping Stats.
Highseq 2500
Manufactured by Illumina
Sourced in United States, Germany
The Illumina HiSeq 2500 is a high-throughput DNA sequencing platform. It is capable of generating large volumes of sequencing data with high accuracy. The HiSeq 2500 uses sequencing-by-synthesis technology to perform rapid, cost-effective, and scalable DNA sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Nextseq 500, by Illumina (3 mentions)
Bioanalyzer 2100 high sensitivity dna gels, by Agilent Technologies (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Hiseq 2000, by Illumina (2 mentions)
Sequence analysis viewer, by Illumina (2 mentions)
Dneasy blood and tissue kit, by Qiagen (2 mentions)
Truseq sample prep kit v2, by Illumina (2 mentions)
Nextseq platform, by Illumina (2 mentions)
Trueseq rna sample preparation kit, by Illumina (2 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Truseq standard total rna kit, by Illumina (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Glycogen, by Thermo Fisher Scientific (1 mentions)
Unisp2, by Qiagen (1 mentions)
Unisp4, by Qiagen (1 mentions)
Unisp5, by Qiagen (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Cleantag small rna library prep kit, by TriLink (1 mentions)
Nanodrop lite spectrophotometer, by Thermo Fisher Scientific (1 mentions)
41 protocols using highseq 2500
1
Extraction and Sequencing of Ancient Bovid DNA
2
ChIP-seq Library Preparation and Sequencing Analysis
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Libraries were prepared using half total volume of eluted ChIP DNA and NEBNext® DNA Library Prep Master Mix Set and Multiplex Oligos for Illumina® (New England Biolabs). Library quality was assessed using Bioanalyzer 2100 High Sensitivity DNA Gels (Agilent). Libraries were subject to 50-bp single end read sequencing on HighSeq 2500 (Illumina) in rapid run format, and reads were aligned to Human genome hg19 using Bowtie2 (Galaxy v2.2.6) Afgan et al., 2016 (link); Supplementary Table S2 ). ENCODE and BEC-associated blacklist regions (including chrN, chrUn, and chrM) were subtracted from each BAM file prior to downstream analysis using the intersect -v function in bedtools (Galaxy v2.1.0). For visualization, Healthy (n = 3) and Asthma (n = 4) BAM files were merged using samtools (Galaxy), Input BAMs subtracted (-bamCompare), and BigWig’s generated and normalized to reads per kilobase per million mapped reads (RPKM, -bamCoverage, deepTools) (Ramírez et al., 2014 (link)).
3
Transcriptomic Analysis of nckap1l Mutant
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Total RNA from 20 wild-type siblings and 20 nckap1llri35 mutant larvae was collected at 5 dpf using the Qiagen RNeasy mini kit (Qiagen, Cat. 74104) according to the manufacturer’s instructions. cDNA for next-generation sequencing was synthesized by using the TruSeq standard total RNA kit (Illumina, 20020598) according to the manufacturer’s instructions. Using an Illumina HighSeq 2500, a total of 11.5 and 10.5 millions of paired-end 50-bp reads were obtained from wild-type and mutant cDNA, respectively. Raw reads were aligned to the zebrafish genome (GRCz10) using TopHat [27 (link)]. The lri35 mutation was mapped to chromosome 11 by MMAPPR [24 (link)]. The NGS and analyzed data are available in the GEO repository at the NCBI (GSE153386).
4
RNA Extraction and Sequencing Protocol
5
Extracellular Vesicle RNA Purification and Sequencing
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EVs were purified by centrifugation at 100,000× g for 1.5 h at 4 °C. The pellet was lysed with 1000 µL Qiazol, supplemented with 1 µL of a synthetic RNA mixture containing three different synthetic control RNAs (UniSp2, two fmol/mL; UniSp4, 0.02 fmol/mL; UniSp5, 0.0002 fmol/mL; Qiagen, Hilden, Germany). RNA extraction was performed using 200 µL chloroform, and phase separation was achieved by centrifugation for 15 min at 12,000× g at 4 °C. RNA was extracted from the upper aqueous phase and purified on a QIAcube liquid handling robot using the miRNeasy Mini kit (Qiagen) with the following modifications: glycogen (Ambion, Austin, TX, USA) was added to the aqueous phase to a final concentration of 50 mg/mL and precipitated with 750 µL 100% ethanol. Columns were washed twice with RPE buffer and RNA was eluted in a single round in 30 µL nuclease-free water and stored at –80 °C.
Small RNA Libraries were prepared from 2 µL RNA, using the CleanTag Small RNA Library Prep Kit (TriLink Biotechnologies, San Diego, CA, USA) according to the manufacturer’s protocol. cDNA libraries were amplified in 21 PCR cycles. Equimolar amounts were pooled and libraries underwent 50 cycles of single-end sequencing on a HighSeq 2500 (Illumina, San Diego, USA).
Small RNA Libraries were prepared from 2 µL RNA, using the CleanTag Small RNA Library Prep Kit (TriLink Biotechnologies, San Diego, CA, USA) according to the manufacturer’s protocol. cDNA libraries were amplified in 21 PCR cycles. Equimolar amounts were pooled and libraries underwent 50 cycles of single-end sequencing on a HighSeq 2500 (Illumina, San Diego, USA).
6
Transcriptome Analysis of Microbial Samples
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Total RNA was treated with DNase to remove DNA contamination. RNA quality was assessed using NANODROP LITE spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Double-stranded cDNA was generated from amplified RNA using the superscript IITM double-strand cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA), as per manufacturer’s instructions. Library preparation, processing, and sequencing were performed at the SciGenom Labs Private Limited, Kerala, India, using the Illumina HighSeq2500 with paired-end (PE) sequencing. Data was submitted with MG-RAST server [26 (link)]. MG-Rast ID of the data sets is available for O1 with accession number is mgm4653349.3; M1 dataset was provided a Gold ID 0eeec568676d676d343733323034392e33 and is under progress at present; however, it can be accessed upon request to the server.
7
Resequencing of Elite Oil Palm Genomes
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After assembling the draft genome for the elite Dura palm tree D1, we also collected samples from oil palm trees from the major plantation area (i.e. Southeast Asia), its place of origin (i.e. Africa) and Central America for re-sequencing. From the collection of oil palms across the world, we selected 13 trees from three forms (i.e. Dura, Pisifera and Tenera) of the species E. guineensis, which included the elite Dura tree D1 used in generating the draft genome (see details in Supplementary Table S1 ). In addition, we also selected five trees of the Compact palm for re-sequencing, which is a hybrid between the two species E. guineensis, and E. oleifera and expected to be exploited in Southeast Asia in the future. We re-sequenced the genomes of 17 trees using paired-end sequencing using the Illumina HighSeq 2500 as described earlier. The sequencing depth of each whole genome was around 5–8-fold (Supplementary Table S1 ).
8
Genomic Analysis of Daphnia Clones
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Daphnia pulex were collected from North America, Europe (Czech Republic), and Asia (China), whereas all D. pulicaria and D. obtusa isolates were derived from North America (for more details seesupplementary table S1, Supplementary Material online). Individual isolates, DNA extraction, and genome sequencing were performed as described in Maruki et al. (2022) . To maximize the likelihood that each individual would originate from a unique genotype, we collected hatchlings in the early spring before the occurrence of subsequent reproduction. Individual isolates were clonally maintained in the laboratory for three generations, and DNA was extracted from 96 isolates per population. The library for each sample was prepared using a Bioo/Nextera kit, followed by tagging with unique oligomer barcodes. Samples from the same population were pooled for sequencing using the Illumina NextSeq 500 or HighSeq 2500 platform, 100 or 150 bp paired-end short reads were generated for each sample. We also included 32 obligately asexual clones sequenced by Tucker et al. (2013) (link) and Xu et al. (2015) (link) into our analysis.
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Daphnia pulex were collected from North America, Europe (Czech Republic), and Asia (China), whereas all D. pulicaria and D. obtusa isolates were derived from North America (for more details see
9
Transcriptomic Analysis of Rapamycin Effects
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For transcriptomic analysis, guts were dissected from control and rapamycin-treated females at the age of 10 days, 30 days, and 50 days. Total RNA was extracted from 25 guts (three replicates) using Trizol (Thermo Fisher) following standard protocols. DNA concentrations were evaluated using a Qubit 2.0 fluorometer (Life Technologies) before DNase I treatment (Thermo Fisher). After adjusting final RNA concentration to 100 ng/µl, 2–3 µl ERCC ExFold RNA Spike-In Mixes (Life Technologies) was added for normalisation to the DNA content of the sample. Ribosomal RNA depletion libraries were generated at the Max Planck Genome Centre Cologne (MPGCC). RNA sequencing was performed with an Illumina HighSeq2500 with 150 bp read length read at MPGCC. At least 37.5 million single-end reads were obtained for each sample.
10
Exome Sequencing for Variant Discovery
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Exome sequencing was performed on proband (III‐1). Sequences were captured through by the Agilent SureSelect version 4 (Agilent Technologies, Santa Clara, CA). The enriched library was sequenced by an Illumina HighSeq2500. The sequencing reads were aligned to the GRCh37.p10 by using BWAv0.59. We then performed local realignment and base quality recalibration of the Burrows–Wheeler aligned reads by using the GATK IndelRealigner and the GATK BaseRecalibrator, respectively. Single‐nucleotide variants and small deletions or insertions were recognized by using the GATK UnifiedGenotyper. The Consensus Coding Sequences Database (20130630) was used to annotate the variants. The obtained variants were filtered and then the variations with minor allele frequencies (MAF) <0.01 were picked.
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