E-MTAB-7105:Sample 1_p
Illumina HiSeq 4000 paired end sequencing; RNA-seq of mouse myeloma cell line SP 2/0 overexpressed with BC094916 against vector control
RNA-seq of mouse myeloma cell line SP 2/0 overexpressed with BC094916 against vector control
Sample 1_p
BC094916 cDNA (General Biosystems, Anhui, China) was cloned into lentiviral vector EGFP-expressing LV201 (Fugene Corp., Guangzhou, China) to generate BC094916 protein. Empty LV201 and BC094916-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 BC094916-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). A. Homogenization with Trizol: 1 mL of TRIzol was added into 1 x 107 cell pellets Invert tube to mix. Place the tube on wet ice temporarily until ready to proceed to 5 Phase Separation, or on dry ice and then move into a -80° freezer for storage of homogenized sample for future extraction.  B. Phase Separation 1. Incubate the homogenized sample for 5 minutes at room temperature. (If tubes are frozen, thaw in a bucket of wet ice before proceeding to room temperature incubation.) 2. In a chemical fume hood, add 0.2 mL of chloroform per 1 mL of TRIzol reagent. 3. Cap sample tubes securely and shake vigorously by hand for 15 seconds. Incubate the samples at room temperature for 3 minutes. 4. Following the incubation, centrifuge the samples at 11,600 x g for 15 minutes at 4 degrees celsius. 5. Following centrifugation, the mixture separates into a lower red phenolchloroform phase, an interphase and a colorless, upper aqueous phase. RNA remains exclusively in the aqueous phase (which is about 60% of the volume of TRIzol reagent used for initial homogenization). However, the organic phase can be saved for subsequent DNA and protein extraction.  C. RNA Precipitation 1. Add 0.5 mL of isopropanol per 1 mL of TRIzol reagent originally used to a new tube. 2. Transfer the aqueous phase to the labeled isopropanol tube. Precipitate the RNA from the aqueous phase by pipetting up and down gently. (Do not vortex.) 3. Incubate the samples for 10 minutes at room temperature, then centrifuge at 11,600 x g for 10 minutes at 4 degrees celsius. 4. The RNA precipitate forms a translucent gel-like pellet on the side and bottom of the tube.  D. RNA Wash 1. Decant the supernatant to a clean, labeled 1.5 mL microcentrifuge tube and set aside. 2. Wash the RNA pellet once with 75% ice-cold ethanol. Use 1 mL of 75% cold ethanol per 1 mL of TRIzol reagent used. 3. Mix by inversion. Do not vortex. Centrifuge at 10,000 x g for 5 minutes at 4 degrees celsius. E. Resuspending the RNA 1. Remove supernatant with a pipet. Quickly spin in a capsule centrifuge to collect any remaining 75% ethanol to the bottom. Remove as much of the remaining ethanol with a pipet and air dry the RNA pellet by leaving the tubes open on the counter for approximately 15-30 minutes. When the pellet is dry, there must be no visible ethanol in the tube. Do not over dry the pellet as it may be difficult to redissolve. 2. Resuspend the RNA pellet in 50 µL of nuclease-free water by pipetting up and down gently and incubating in a 60 degrees celsius water bath for 10 minutes (can incubate longer if necessary, up to one hour). Flick (do not vortex) gently to mix and quickly spin to collect the solution.  F. DNase Treatment of RNA 1. Add 0.1 volume (5.6 µL) of 10X DNase I buffer and 1 µL of DNase I (2 units) to the RNA. Mix gently by flicking tube (DO NOT VORTEX) and incubate in a 37oC water bath for 20 minutes. 2. Add 0.1 volume of DNAse Inactivation Reagent (6.17 µL) to the RNA. (Make sure to vortex the DNAse Inactivation Reagent before addition to the RNA.) Flick (do not vortex) the RNA tube to disperse the reagent. Incubate for 2 minutes at room temperature. Flick the tube once more during the incubation to redisperse the DNase Inactivation Reagent. 3. Centrifuge at 10,000 x g for 1 minute at 4 degrees celsius. Transfer the supernatant RNA solution to a 1.5 mL micrewtube labeled with the BPC number, patient initials, and parent ID or barcode.  G. Determination of RNA Concentration Quantitate RNA by measuring absorption at 260 nm – refer to the Quantitation of Nucleic Acids procedure, GEN-2.  Libraries were prepared using the TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold (Illumina).
Beijing Institute of Basic Medical Sciences
E-MTAB-7105:Sample 1_p
Experimental Factor: genotype
LV201-eGFP