The Twist Library Preparation EF 2.0 protocol was performed on a Perkin Elmer Sciclone robot with 100 ng DNA and 7 PCR cycles. In total, 1536 different indexes were used. After the library preparation, the DNA concentrations were measured with nanodrop and 16 samples with 200 ng DNA of each sample were pooled for the hybridization step. The pooled DNA was dried using vacuum pressure. To each dried sample, 4 ul of the mtDNA probe panel (Twist) was added and hybridized in a PCR machine at 70C for 17 hours. The capture of the mtDNA was performed on a Perkin Elmer robot with the Twist Target enrichment standard hybridization v2 protocol. For each pool of samples, 15 PCR cycles were performed. DNA concentrations were measured with a fluorescence-based assay and size distribution was checked with the Tapestation system. Sequencing was performed at 2 × 150 bp on an Illumina Novaseq6000 system at up to 10,000x per sample.
Sciclone robot
Manufactured by PerkinElmer
The Sciclone robot is a liquid handling system designed for automated sample preparation and processing in the laboratory. It is capable of precise and accurate liquid dispensing, dilution, and transfer operations. The Sciclone robot is suitable for a variety of applications, including high-throughput screening, sample preparation, and assay development.
Automatically generated - may contain errors
2 protocols using sciclone robot
1
mtDNA Enrichment and Sequencing Protocol
2
RNA-seq Protocol with Qiagen AllPrep
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Samples were extracted using the AllPrep DNA/RNA Mini Kit (Qiagen) on a Qiacube robot. RNA quality was tested by running the samples on a Bioanalyzer 2100 from Agilent, using the RNA6000 Nano LabChip kit (Agilent Technologies, Inc), and cDNA libraries were prepared on Sciclone robot (PerkinElmer Inc) using the RNATRUSEQ protocol (Illumina Inc). RNAseq was performed using Hiseq 2000 with 100 bp paired‐end reads. Samples with low sequencing throughput (<10 M reads) were removed from the analysis. The selected RNAseq samples were aligned with the GEMTools RNAseq pipeline v1.7 (http://gemtools.github.io ).15 The transcriptome was generated from version 15 of the Gencode annotation. After mapping, all alignments were filtered to increase the number of uniquely mapping reads. The filter criteria contained a minimum intron length of 20, a minimum exon overlap of 5 and a filter step against the reference annotation checking for consistent pairs and junctions where both sides align to the same annotated gene. Quantifications and read counts were calculated using the Flux Capacitor15 to create gene‐level read counts that were used for the differential expression analysis.
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