Amplicons of the V4 hypervariable regions of the 16S rRNA gene were generated according to the Earth Microbiome Protocol140 (link). In brief, 10 μg of extracted DNA from each sample was used as template for triplicate PCR reactions utilizing individually barcoded 515F forward primers (GTGYCAGCMGCCGCGGTAA) with Illumina adapters and the 806R reverse primer (GGACTACNVGGGTWTCTAAT) with Illumina adapters. Triplicates were combined and measured using the Qubit 3.0 system with Broad-Range DNA Reagents (Thermo Fisher Scientific). Samples were pooled in equimolar concentrations and sent out for 2x250 paired-end sequencing utilizing an Illumina MiSeq system at the University of Rhode Island. We obtained a total of 3,806,054 quality-filtered sequences across all 90 samples. The average sequencing depth was 41,509 reads in the control group and 44,169 in the MDD group. Sequences can be found at the NCBI Short Read Archive under BioProject ID PRJNA591924.
Adapters
Manufactured by Illumina
Sourced in United States
Illumina adapters are oligonucleotide sequences that are ligated to DNA fragments during library preparation for Illumina sequencing platforms. They serve as priming sites for sequencing reactions and enable the identification of individual samples in multiplexed sequencing runs.
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Lab products found in correlation
Bioruptor pico sonication system, by Diagenode (4 mentions)
Qiaamp dna blood mini kit, by Qiagen (4 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
Nextseq 550, by Illumina (3 mentions)
Rneasy kit, by Qiagen (3 mentions)
Miseq system, by Illumina (3 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Truseq rna sample preparation kit, by Illumina (2 mentions)
Hiseq 2000 platform, by Illumina (2 mentions)
Sciclone ngsx workstation, by PerkinElmer (2 mentions)
Quant it dsdna assay, by Thermo Fisher Scientific (2 mentions)
Agilent 2100 bioanalyzer results, by Agilent Technologies (2 mentions)
Truseq stranded mrna kit, by Illumina (2 mentions)
Pippin prep, by Sage Science (2 mentions)
Truseq rna sample preparation kit v2, by Illumina (2 mentions)
Hiseq 2000, by Illumina (2 mentions)
Qiaamp dna blood extraction kit, by Tiangen Biotech (2 mentions)
Miseq 2000 sequencing platform, by Illumina (2 mentions)
Miseq platform, by Illumina (2 mentions)
Agencourt ampure xp bead, by Beckman Coulter (2 mentions)
41 protocols using adapters
1
16S rRNA Amplicon Sequencing Protocol
2
Foxtail Millet Transcriptome and Methylome Analysis
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The RNA-seq data of four tissues namely, root (SRX128223), stem (SRX128225), leaf (SRX128224) and spica (SRX128226), and a drought stress library (SRR629694) as well as control (SRR629695) were retrieved from European Nucleotide Archive (http://www.ebi.ac.uk/ena ). The reads were processed to generate RPKM following Mishra et al.16 (link) and heat map was displayed using MeV v4.933 (link).
Total genomic DNA of foxtail millet cultivars ‘IC-403579’ and ‘IC-480117’ were sonicated and the fragmented DNA was end-repaired and ligated with adapters following manufacturer’s instructions (Illumina, San Diego, CA). Sodium bisulfite treatment was given to purified DNA fragments and the sample was PCR amplified using adapter specific primers. The amplified DNA was used to prepare library and sequenced by Illumina Genome Analyzer (GAIIx) according to manufacturer’s instructions. Raw reads were analysed using Bismark tool40 (link).
Total genomic DNA of foxtail millet cultivars ‘IC-403579’ and ‘IC-480117’ were sonicated and the fragmented DNA was end-repaired and ligated with adapters following manufacturer’s instructions (Illumina, San Diego, CA). Sodium bisulfite treatment was given to purified DNA fragments and the sample was PCR amplified using adapter specific primers. The amplified DNA was used to prepare library and sequenced by Illumina Genome Analyzer (GAIIx) according to manufacturer’s instructions. Raw reads were analysed using Bismark tool40 (link).
3
Transcriptome analysis of young leaves
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Young leaves were collected with two biological replicates at seedling stage from the field and immediately frozen in liquid nitrogen and stored at −80°C until use. Total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer's instructions. The quality of purified RNA was initially evaluated on agarose gel and then quantified using NanoDrop spectrophotometer (Thermo Fisher Scientific, Inc.). The integrity of RNA samples were further evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.). The TruSeq TM RNA Sample Preparation Kit (Illumina, Inc.) was then used according to the manufacturer's instructions, to construct cDNA libraries. Concisely, poly-A mRNA was purified and fragmented into short fragments and used as templates for first strand cDNA synthesis. Then DNA polymerase I and RNase H were used to synthesize the second-strand cDNA. Purified short double strand cDNA fragments were connected with adapters (Illumina). Suitable ligated cDNA fragments were selected as templates for the PCR amplification for the finally library construction. Finally, the cDNA libraries were sequenced using Illumina HiSeq 2000 sequencing platform at National Key Laboratory for Crop Genetic Improvement in Huazhong Agricultural University, Wuhan, China.
4
Illumina HiSeq® 2500 RNA Sequencing Library Prep
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The TruSeq™ RNA Sample Preparation Kit (Illumina, Inc.) was used to construct cDNA libraries in accordance with the manufacturer’s instructions. Briefly, poly-A mRNA was purified and fragmented into short fragments and used as templates for first-strand cDNA synthesis. DNA polymerase I and RNase H were used to synthesize the second-strand cDNA. Purified short double-strand cDNA fragments were connected with adapters (Illumina). Suitable ligated cDNA fragments were selected as templates for PCR amplification for final library construction. Finally, the cDNA libraries were sequenced using the Illumina HiSeq® 2500 platform.
5
RNA-Seq Data Processing Pipeline
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Raw reads quality was visually inspected by means of FastQC software68 and then processed to remove low quality bases and contaminants (Illumina adapters). Cleaning phase was then performed with Trimmomatic software version 0.3669 (link) using an average quality cut-off of 30 (Phred score) and a minimum read length of 40 bp. Cleaned reads were then mapped against predicted mRNAs (PN40024 12X v2 grape reference transcriptome) obtained from the gene prediction version 2.0 of the National Centre for Biotechnology Information. Mapping was performed using Bowtie270 (link) tool with default parameters. Alignments were first converted in a binary alignment map (BAM), a binary representation of the Sequence Alignment/MAP (SAM), and then sorted and indexed for the count of reads per mRNA, using SAMtools71 (link) software package. The number of reads aligning to each transcript were counted using an ad hoc Python script.
6
DNA Library Preparation and Sequencing
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A previously described procedure was followed for library construction and sequencing [22 (link)]. Briefly, DNA was sheared (Covaris), end repaired (Lucigen), polyadenylated (Lucigen), and ligated to adapters (Illumina) for paired-end data generation. DNA sequencing was performed on the Illumina Genome Analyzer II and generated between 114 and 260 Gbp of sequence data for each tissue studied and haploid coverage ranging from 29.24 to 72.17 (S2 Table ).
7
Targeted Sequencing of Eye Disease Genes
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For sequencing, 1 μg of the DNA sample from the proband of each family was sheared into fragments that were 200–500 bp long. The sheared fragments received blunt-end repair, and Klenow exonuclease was used to add a single-adenine base to the 3′ ends. Then, adapters (Illumina, San Diego, CA) were ligated to the repaired ends, and the DNA fragments were amplified in a PCR after ligation. PCR conditions were: denaturing at 98 °C for 2 min followed by 8 cycles of 98 °C for 30 s, 65 ℃ for 30 s and 72 °C for 1 min, then a final extension step at 72 °C for 10 min. The targeted DNA was captured using a customized panel of 762 genes, which included all the known genes related to eye diseases and sequenced by the Illumina HiSeq X Ten machine [9 (link)]. All the genes we detected are listed in Appendix 1.
8
RNA Extraction and cDNA Library Preparation Protocol
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Total RNA was extracted from mixed leaf tissue samples from four individual plants using RNAiso reagent (TaKaRa Shuzo Co. Ltd., Japan) according to the manufacturer’s instructions. The integrity and purity of the RNA were examined by agarose gel electrophoresis and measured with a NanoDrop 8000 spectrophotometer (Thermo Scientific, United States).
High-quality RNA from each sample was used for library construction following the manufacturer’s protocol (Illumina, United States). Briefly, oligo (dT) magnetic beads were used to obtain purified poly-A mRNA, which was fragmented into small pieces in fragmentation buffer. These small fragments were used as templates for first-strand cDNA synthesis with random hexamer primers. Second-strand cDNA was synthesized using DNA polymerase I, RNase H, dNTP, and buffer. Short cDNA fragments were purified with a QiaQuick PCR extraction kit. Following end repair and the addition of poly(A), the cDNA fragments were attached to adapters (Illumina). After selecting suitable ligated cDNA fragments, PCR amplification was performed and the PCR products were purified. The cDNA libraries were sequenced on the Illumina HiSeq™ 2000 platform.
High-quality RNA from each sample was used for library construction following the manufacturer’s protocol (Illumina, United States). Briefly, oligo (dT) magnetic beads were used to obtain purified poly-A mRNA, which was fragmented into small pieces in fragmentation buffer. These small fragments were used as templates for first-strand cDNA synthesis with random hexamer primers. Second-strand cDNA was synthesized using DNA polymerase I, RNase H, dNTP, and buffer. Short cDNA fragments were purified with a QiaQuick PCR extraction kit. Following end repair and the addition of poly(A), the cDNA fragments were attached to adapters (Illumina). After selecting suitable ligated cDNA fragments, PCR amplification was performed and the PCR products were purified. The cDNA libraries were sequenced on the Illumina HiSeq™ 2000 platform.
9
Bacterial 16S rRNA Amplicon Sequencing
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The next-generation sequencing analysis was based on the 16S rRNA gene of bacteria. The V3-V4 regions of the gene were amplified using universal region-specific primers 341F (5′-CCT AYG GGR BGC ASC AG-3′) and 806R (5′-GGA CTA CNN GGG TAT CTA AT-3′) (Novogene, Singapore) tagged with sample-identifying barcodes. PCR products of the proper size were selected by 2% agarose gel electrophoresis. The DNA fragments were end-repaired and A-tailed then further ligated with Illumina adapters. The libraries were generated and sequenced on a paired-end Illumina platform (Novogene). The sequences have been deposited to NCBI under the accession number PRJNA919511.
10
Targeted Sequencing of Genomic DNA from Bone Marrow
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Genomic DNA was extracted from bone marrow using a QIAamp DNA Blood Mini Kit (Qiagen, Venlo, The Netherlands). Approximately 1.5 μg of genomic DNA was fragmented to segments between 150 and 250 bp in length using the Bioruptor Pico Sonication System (Diagenode, Belgium). The resulting DNA was then end-repaired and ligated to Illumina adapters (Illumina, San Diego, CA, USA). Sequence indexes were added to the samples to allow all samples to be sequenced in a single flow cell. Small fragments of ~100 bp and unligated adapters were removed using the AMPure purification system (Agencourt Bioscience, Beverly, MA, USA). Sequencing libraries were then hybridized with the capture probes. Streptavidin-coated paramagnetic beads were used to remove unbound DNA. The captured DNA was finally eluted from the magnetic beads by digestion of the cRNA capture probes and purified. The enriched DNA was then amplified using universal primers targeting the paired-end adapters, clusters were generated, and DNA was sequenced on a NextSeq 550 instrument (Illumina) with 2×151 bp reads. All procedures were performed according to the manufacturer’s instructions.
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