Bioanalyzer 2100 dna chip 7500

RNA was extracted from SCLC cells (H69 and H69AR). The RNA quality was assessed with a Bioanalyzer 2100 DNA Chip 7500 instrument (Agilent Technologies), and samples with an RNA integrity number (RIN) of over 7 were further analysed by RNA-seq. All sequencing reactions were performed on an Illumina HiSeq 2000 instrument (Illumina, San Diego, CA, USA). We used HISAT2 (version 2.1.0) with the default setting to map the RNA-seq data to the human reference genome (NCBI38/hg38). We aggregated the read counts at the gene level using HTSeq. The R package ‘edgeR’ was used to analyse the difference in gene expression data (raw count), and the R package ‘complexHeatmap’ was used to visualize the results [30 (link), 31 (link)].

For capture enrichment, pools of libraries (~104 ng/library and 48 libraries/pool) were prepared, and 5.0 ug/pool was used for in-solution hybridization to custom capture a design [150825_HG19_10bp_Mende_chr_EZ_HX3] that covered all exons and 10 bp intron-exon boundary regions on each side of the exons of the 47 genes (Nimblegen).
Human COT1 DNA and full-length blocking oligonucleotides (Sigma-Aldrich Inc., St. Louis, MO, USA) were added into the hybridization to block repetitive genomic sequences and the adaptor sequences. Hybridization was done at 56 °C O/N. Post-capture LM-PCR amplification was performed using the Library Amplification Readymix containing KAPA HiFi DNA Polymerase (Kapa Biosystems, Inc., Cat # KK2612) with 12 cycles of amplification. After the final AMPure XP bead purification, quantity and size of the capture library was analyzed using an Agilent Bioanalyzer 2100 DNA Chip 7500. The enriched library pools (48 libraries/pool or 96 per lane) were sequenced on a HiSeq 2000 to generate 2 × 100 bp reads. On average, for these 134 samples, including 47 samples that were whole genome amplified, 397 Mb of mapped sequence data (295 Mb unique with 537.38× sequence coverage) across the length of the targeted region were generated. On average, 96% of bases in the design were covered at a ≥ 20× depth.

Genomic DNA was isolated from overnight cultures using a Wizard Genomic DNA purification kit (Promega, Madison, WI) and quantified with PicoGreen (Molecular Probes, Eugene, OR). Sequencing libraries were prepared using the Illumina Paired-End Sample Preparation Kit according to the manufacturer's protocol but with the recommended modification of performing gel extraction incubations at room temperature [35] (link). Prior to sequencing, library quality and insert size were evaluated on an Agilent Bioanalyzer 2100 DNA 7500 chip. Libraries were sequenced at the Iowa State University DNA Facility on an Illumina GAIIx for either 75 cycles (strains SW114, 12939, 84-15995, H465, and D74) or 100 cycles (strains Nagasaki, MN-H, 29755, 174 and SW140). GenBank accession numbers for the genomes are given in Table 2 and Kuehn et al. [36] (link).