ena-STUDY-ROSWELL PARK CANCER INSTITUTE-20-11-2019-03:17:21:414-474
Whole exome and transcriptome sequencing of human lung cancer tumors
Five freshly resected human primary tumors with non-small cell lung cancer (adenocarcinoma or squamous cell carcinoma) were dissociated into single-cell suspensions using an enzyme cocktail and frozen. EpCAM-positive, EpCAM-negative/CD45-positive, and EpCAM-negative/CD14-positive groups of cells were later isolated from thawed cell suspensions using antibody-coated magnetic beads. Total RNA and genomic DNA were simultaneously extracted from the three groups of cells as well as from the thawed tumor cell suspensions and frozen tumor-adjacent lung tissue using Qiagen AllPrep DNA/RNA Mini Kit. Nucleic acid preparations were quantified with Qubit assay.  Eighty to 100 ng of DNA/RNA was used to generate sequencing libraries using Agilent SureSelect XT Low Input Human All Exon V7 (with 9 pre-capture and 10 post-capture PCR cycles) and KAPA RNA HyperPrep Kit with RiboErase (HMR) (with 15 PCR cycles) kits respectively for DNA and RNA. For library indexing, Agilent SureSelect XT Low Input Unique Dual and IDT for Illumina TruSeq UD Indexes respectively were used for DNA and RNA. Libraries were quantified with Agilent Tapestation 4200 and KAPA Biosystems qPCR quantitation All of the RNA/DNA sequencing libraries were together subjected to sequencing to obtain paired 101 b reads on Illumina NovaSeq 6000 platform with single flow cell (350 pM each library; 1% PhiX library spike-in) and Illumina NovaSeq 6000 S1 Reagent kit (200 cycles). Demultiplexed sequencing data, with adapters trimmed with bcl2fastq (v 2.20) based on 90% sequence identity threshold, are provided here. RNA sequencing failed for the library for the thawed tumor cell suspension of patient 63.
Five freshly resected human primary tumors with non-small cell lung cancer (adenocarcinoma or squamous cell carcinoma) were dissociated into single-cell suspensions using an enzyme cocktail and frozen. EpCAM-positive, EpCAM-negative/CD45-positive, and EpCAM-negative/CD14-positive groups of cells were later isolated from thawed cell suspensions using antibody-coated magnetic beads. Total RNA and genomic DNA were simultaneously extracted from the three groups of cells as well as from the thawed tumor cell suspensions and frozen tumor-adjacent lung tissue using Qiagen AllPrep DNA/RNA Mini Kit. Nucleic acid preparations were quantified with Qubit assay.  Eighty to 100 ng of DNA/RNA was used to generate sequencing libraries using Agilent SureSelect XT Low Input Human All Exon V7 (with 9 pre-capture and 10 post-capture PCR cycles) and KAPA RNA HyperPrep Kit with RiboErase (HMR) (with 15 PCR cycles) kits respectively for DNA and RNA. For library indexing, Agilent SureSelect XT Low Input Unique Dual and IDT for Illumina TruSeq UD Indexes respectively were used for DNA and RNA. Libraries were quantified with Agilent Tapestation 4200 and KAPA Biosystems qPCR quantitation All of the RNA/DNA sequencing libraries were together subjected to sequencing to obtain paired 101 b reads on Illumina NovaSeq 6000 platform with single flow cell (350 pM each library; 1% PhiX library spike-in) and Illumina NovaSeq 6000 S1 Reagent kit (200 cycles). Demultiplexed sequencing data, with adapters trimmed with bcl2fastq (v 2.20) based on 90% sequence identity threshold, are provided here. RNA sequencing failed for the library for the thawed tumor cell suspension of patient 63.