title: Illumina HiSeq 4000 paired end sequencing; RNA-seq of mouse myeloma cell line SP 2/0 overexpressed with Gm614 against vector control
design: RNA-seq of mouse myeloma cell line SP 2/0 overexpressed with Gm614 against vector control
library_construction_protocol: Stable GM614- and EGFP-expressing SP 2/0 cells, and the control EGFP-expressing SP 2/0 cells were collected after after cultured for 2 days in fresh medium. Samples were washed twice with PBS and 1 mL of TRIzol per 1 x 107 cell pellets was added and frozen in -80℃. GM614 cDNA (General Biosystems, Anhui, China) was cloned into lentiviral vector EGFP-expressing LV201 (Fugene Corp., Guangzhou, China) to generate GM614 protein. Empty LV201 and GM614-expressing LV201 was transfected into SP 2/0 cells (ATCC® CRL-1581™, https://www.atcc.org/products/all/CRL-1581.aspx), and stable transfectants were identified by EGFP expression and drug selection (Puromycin, Sigma, 10 μg/ml). Stable GM614-expressing SP 2/0 cells and LV201-transduced SP 2/0 cells were cultured in fresh ATCC-formulated Dulbecco's Modified Eagle's Medium (Catalog No. 30-2002) containing 10% FBS, 2 mM glutamine, penicillin (100 IU/ml), streptomycin (100 ug/ml), and 50 mM 2-mercaptoethanol. Cells were collected on ice after cultured for 2 days in fresh medium. Data were aligned using STAR with options --outFilterScoreMinOverLread 0.4 --outFilterMatchNminOverLread 0.4, against the C3H/HeJ genome as a reference (ftp://ftp.ensembl.org/pub/release-89/fasta/mus_musculus_c3hhej/dna/Mus_musculus_c3hhej.C3H_HeJ_v1.dna_sm.toplevel.fa.gz).  Total RNA from cells was isolated and purified using the RNeasy Mini Kit (Qiagen, Venlo, Netherlands). All RNA content in the samples was measured with a NanoDrop®ND-1000 spectrophotometer, and the RNA quality was assessed using RNA 6000 NanoChips with the Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA). Libraries were prepared using the TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold (Illumina).
attributes.tag: Experimental Factor: genotype
attributes.value: empty vector LV201
attributes.tag: Experimental Factor: phenotype
attributes.value: empty vector