Illumina HiSeq 4000 paired end sequencing; RNAseq analysis of SCLC PDX models
RNAseq analysis of SCLC PDX models
We performed RNA sequencing of SCLC PDXs. Patient samples 46 samples from the 33 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 and CDX 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). 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.