The data and methods used have been described previously [54 (link)]. Briefly, two quail lines were used, divergently selected on their sociability [19 (link)]: high social (HSR) and low social (LSR) reinstatement behaviour. A total of 10 individuals from generation 50 of each quail line were sequenced after equimolar DNA pooling. Sequencing was performed (paired-ends, 100 bp) on a HiSeq 2000 sequencer (Illumina), using one lane per line (TruSeq sbs kit version 3). The reads (190,159,084 and 230,805,732 reads, respectively, for the HSR and LSR lines) were mapped to the CoJa2.2 genome assembly using BWA [112 (link)], with the mem algorithm. Data are publicly available under SRA accession number SRP047364. Within each line, the frequency of the reference allele was estimated for all SNPs covered by at least 5 reads, using Pool-HMM [113 (link)]. This analysis provided 13,506,139 SNPs with allele frequency estimates in the two lines. FLK values [55 (link)] were computed for all these SNPs, and the local score method [54 (link)] was applied to the p value on single-marker tests.
Truseq sbs kit version 3
Manufactured by Illumina
The TruSeq SBS kit version 3 is a reagent kit designed for use with Illumina sequencing platforms. It provides the necessary reagents for performing sequencing-by-synthesis (SBS) reactions. The kit includes all the required components to generate high-quality sequence data from DNA samples.
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Lab products found in correlation
Hiseq 2000, by Illumina (3 mentions)
Hiseq 2000 sequencer, by Illumina (2 mentions)
Truseq nano dna library preparation kit, by Illumina (2 mentions)
Truseq rna sample prep kit v2, by Illumina (1 mentions)
Truseq pe cluster kit v3 cbot hs, by Illumina (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Mirvana paris total rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Picofrit column 9, by New Objective (1 mentions)
Dionex ultimate 3000 rslc lc system, by Thermo Fisher Scientific (1 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Truseq nano dna library, by Illumina (1 mentions)
Hiseq x, by Illumina (1 mentions)
Truseq rna sample preparation kit, by Illumina (1 mentions)
6 protocols using truseq sbs kit version 3
1
Genome-wide selection for social behavior
2
RNA-Seq and miRNA-Seq Analysis Protocol
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RNA sequencing analysis was performed as described [16 ]. Sequencing RNA libraries were prepared according to the manufacturer’s protocol (TruSeq RNA Sample Prep Kit v2, Illumina) and loaded onto flow cells (8-10pM) to generate cluster densities of 700,000/mm2 following the standard protocol for the Illumina cBot and cBot Paired-end cluster kit version-3. Cells were sequenced on an Illumina HiSeq 2000 using TruSeq SBS kit version 3 and HCS v2.0.12 data collection software and data analyzed using the MAPRSeq v.1.2.1 system and the Bioinformatics Core standard tool, which includes alignment with TopHat 2.0.6 [17 (link), 18 (link)] and gene counts with the featureCounts software [19 (link)]. miRNA-Seq data were analyzed using CAP-miRSeq v1.1 [20 (link)] and normalization and differential expression analysis performed using edgeR 2.6.2 [21 (link)]. Gene expression was normalized to 1 million reads and corrected for gene length (reads per kilobasepair per million mapped reads, RPKM), and miRNA expression levels expressed as normalized total reads.
3
Whole Genome and Transcriptome Sequencing
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DNA libraries of the tumor and matched normal samples were prepared using the TruSeq DNA library Preparation Kit Sets A and B (Illumina; estimated insert size of 343 bp) or TruSeq Nano DNA library Preparation Kit (Illumina; estimated insert size of 350 bp). Clusters were generated with cBot and the TruSeq PE Cluster Kit v3 cBot HS (15023336_A, Illumina). Paired-end sequencing was performed on Illumina HiSeq2000 (2x 100 bp), HiSeq2500 (2x125 bp), or Hiseq-X10 (2x 150 bp) instruments using the TruSeq SBS Kit, Version 3 (200 cycles).
RNA libraries of the tumor samples and sorted germinal center B-cells from non-neoplastic tonsils were prepared using the TruSeq RNA library preparation Kit Set A and B, at an insert size of ~300 bp according to manufacturer´s instructions. Two barcoded libraries were pooled per lane and sequenced on Illumina HiSeq2000 or HiSeq2500.
RNA libraries of the tumor samples and sorted germinal center B-cells from non-neoplastic tonsils were prepared using the TruSeq RNA library preparation Kit Set A and B, at an insert size of ~300 bp according to manufacturer´s instructions. Two barcoded libraries were pooled per lane and sequenced on Illumina HiSeq2000 or HiSeq2500.
4
mRNA Sequencing and Proteomic Analysis of MSC-derived EVs
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mRNA was isolated from MSC-derived EVs using the mirVana PARIS total RNA isolation kit (Life Technologies) according to the manufacturer’s protocol. mRNA sequencing was performed at the Mayo Clinic Bioinformatic Core, as previously described13 (link). Samples were sequenced on an Illumina HiSeq 2000 using TruSeq SBS kit version 3 and HCS v2.0.12 data collection software and data analyzed using the MAPRSeq v.1.2.1 system and the Bioinformatics Core standard tool, which includes alignment with TopHat 2.0.627 (link),28 (link) and gene counts with the featureCounts software29 (link). Normalized expression values for each gene were calculated as reads per kilobase per million (RPKM).
In addition, liquid chromatography mass spectrometry (LC-MS/MS) proteomic analysis was performed as previously described30 (link),31 (link). EV pellets were solubilized and lysed, and protein samples denatured. Aliquots were resolubilized in reducing sample buffer and samples electrophoresed. Gel sections were digested with trypsin31 (link), and peptides extracted and transferred onto a PicoFrit column 9 (NewObjective), self-packed with Agilent Poroshell 120 S 2.7 µm EC-C18 stationary phase, using a Dionex UltiMate 3000 RSLC LC system (Thermo-Fisher Scientific). Peptides were separated and eluting peptides analyzed using a QExactive mass spectrometer (Thermo-Fisher Scientific).
In addition, liquid chromatography mass spectrometry (LC-MS/MS) proteomic analysis was performed as previously described30 (link),31 (link). EV pellets were solubilized and lysed, and protein samples denatured. Aliquots were resolubilized in reducing sample buffer and samples electrophoresed. Gel sections were digested with trypsin31 (link), and peptides extracted and transferred onto a PicoFrit column 9 (NewObjective), self-packed with Agilent Poroshell 120 S 2.7 µm EC-C18 stationary phase, using a Dionex UltiMate 3000 RSLC LC system (Thermo-Fisher Scientific). Peptides were separated and eluting peptides analyzed using a QExactive mass spectrometer (Thermo-Fisher Scientific).
5
Illumina TruSeq Nano DNA Library Preparation
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The DNA libraries of the tumor and matched control samples were prepared according to the Illumina TruSeq Nano DNA Library protocol using the TruSeq Nano DNA Library Preparation Kit (Illumina, Hayward, CA; estimated insert size of 350 bp). Paired-end sequencing was performed on Illumina HiSeq X (2 × 150 bp) instruments using the TruSeq SBS kit, Version 3.
6
RNA Sequencing Protocol for Illumina HiSeq 2000
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The RNA was sequenced by a commercial provider that prepared cDNA libraries using a TruSeq RNA sample preparation kit (Illumina, San Diego, CA), generating about 19 million 100-bp reads. The libraries were quantified by real-time PCR and sequenced (2 × 100 paired-end reads) on a HiSeq 2000 sequencer (Illumina) using the TruSeq SBS Kit version 3 (Illumina).
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