Illumina HiSeq 4000 paired end sequencing; RNAseq analysis of meso PDX models
RNAseq analysis of meso PDX models
Sample 13_p
We performed RNA sequencing of meso PDXs. Patient samples 17 samples from the 17 patients reported in this study were collected with informed consent from patients under protocols approved by the MSKCC (MSK IMPACT Protocol 12-245; PDX Protocol 14-091; Biospecimen Protocol 06-107). Metadata for each patient can be found in Supplementary Table 1.   Patient-derived xenografts All animal experiments were approved by the Memorial Sloan Kettering Cancer Center (MSKCC) Animal Care and Use Committee and mice were housed in accredited facilities under pathogen-free conditions. PDX models were generated from primary tumors and whole blood samples as described previously (Daniel, V. C. et al. Cancer Res 69, 3364-3373, doi:10.1158/0008-5472.CAN-08-4210 (2009). Xenograft protocol as follows: Generation and maintenance of primary xenografts and cell lines. Over an 18-mo period, discarded tissues from three chemo-naive SCLC patients undergoing therapeutic bronchoscopy for acute bronchial obstruction were obtained fresh and transported to the laboratory in 1× PBS at 4°C. All samples were anonymized and obtained in accordance with the Johns Hopkins University Institutional Review Board. Due to the small amount of material available, the entire sample was used to generate a xenograft. Under aseptic conditions, tumor samples were finely minced with razor blades, vigorously triturated in 1× PBS, passed through a 60-μm filter, centrifuged, and then resuspended in 500 μL of Matrigel (BD Biosciences) at 4°C. Cells were then injected s.c. in the flanks of five nonobese diabetic/severe combined immunodeficient mice that were monitored for tumor growth. When the P0 tumors reached 1 cm in diameter, the mouse was sacrificed and the tumor divided into sections for snap freezing, frozen tissue sectioning, formalin fixation, conventional cell culture, or serial passage. All animal studies were done in accordance with protocols approved by the Johns Hopkins University Animal Care and Use Committee. Serial passage in vivo was done by disaggregating the tumor as described above. Aliquots of cells were then injected into the flanks of athymic nude mice in Matrigel or cryopreserved in 90% RPMI (Invitrogen)/10% DMSO (Sigma). Conventional cell lines were established by seeding an aliquot of disaggregated cells in culture with advanced RPMI (Invitrogen)/1% bovine calf serum (Invitrogen). Cell lines were passaged and cryopreserved in standard fashion for SCLC cultures. Publicly available SCLC cell lines were obtained from American Type Culture Collection (ATCC) and cultured in advanced RPMI (Invitrogen)/1% bovine calf serum. Xenografts derived from these conventional cell lines were grown in the flanks of nude mice as described above. Orthotopic xenografts were generated by dorsoscapular, transcutaneous injection of cells suspended in Matrigel into the right lung of nude mice, essentially as described RNA extraction and sequencing was done in collaboration with Genewiz. Total RNA was extracted from fresh frozen cell pellet samples using Qiagen RNeasy Plus Universal mini kit following manufacturer's instructions (Qiagen, Hilden, Germany). Extracted RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA). RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina following manufacturer's instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were first enriched with Oligo(dT) beads. Enriched mRNAs were fragmented for 15 minutes at 94C. First strand and second strand cDNAs were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3'ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by limited-cycle PCR. The sequencing libraries were validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). Sample preparation for RNA sequencing and subsequent analysis was performed as in 26. Briefly, Illumina HiSeq instrument (4000 or equivalent; a 2x150bp Paired End (PE)) according to manufacturer's instructions was used for RNA sequencing in collaboration with Genewiz.