NextSeq 500 sequencing; RNA-seq to investigate YBX3 siRNA knockdown in HeLa cells
RNA-seq to investigate YBX3 siRNA knockdown in HeLa cells
siCtrl_LM_3_s
Adherent HeLa cells were grown at 37C and 5% CO2 in DMEM, supplemented with 10% FBS Gold (PAA), 1% Penicillin/Streptomycin (Sigma) and 1% L-glutamine (Gibco), until 70-80% confluent.   Cells were trypsinized, counted (Biorad TC20) and 200,000 cells were seeded per well in a 6-well dish for reverse transfection of siRNAs following the manufacturer s recommendations (Lipofectamine RNAiMax, Invitrogen).    YBX3 and control siRNAs were obtained from Dharmacon (YBX3 pool: SMARTpool ON-Targetplus L-015793-00 or YBX3 3UTR targeting: ON-Targetplus L-015793-07; and control pool: ON-Targetplus non-targeting siRNAs 1-4).     The siRNA used to target the YBX3 3UTR was YBX3 smartpool J-015793-07. Cells were harvested 48 hours after transfections. All siRNA sequences are: YBX3 smartpool J-015793-05 (Sense: 5-GGAAGGGAGUCGUUACGCU-3, Antisense: 5-AGCGUAACGACUCCCUUCC-3),      YBX3 smartpool J-015793-06 (Sense: 5-GAUGAAGGAUGGAGUCCCA-3, Antisense: 5-UGGGACUCCAUCCUUCAUC-3), YBX3 smartpool J-015793-07 (Sense: 5-GCAAUAAAGUGGAAGACUA-3, Antisense: 5-UAGUCUUCCACUUUAUUGC-3),   ON-Targetplus YBX3 J-015793-08 (Sense 5-GCACGGAAGACAAGAGAGU-3, Antisense: 5??? ACUCUCUUGUCUUCCGUGC-3), ON-Targetplus Non-targeting #1 D-001810-01 (Sense: 5??? UGGUUUACAUGUCGACUAA-3, Antisense:  5-UUAGUCGACAUGUAAACCA-3),    ON-Targetplus Non-targeting #2 D-001810-02 (Sense: 5??? UGGUUUACAUGUUGUGUGA-3, Antisense: 5-UCACACAACAUGUAAACCA-3),     ON-Targetplus Non-targeting #3 D-001810-03 (Sense: 5??? UGGUUUACAUGUUUUCUGA-3, Antisense: 5-UCAGAAAACAUGUAAACCA-3),      ON-TARGETplus Non-targeting #4 D-001810-04 (Sense: 5??? UGGUUUACAUGUUUUCCUA-3, Antisense: 5-UAGGAAAACAUGUAAACCA -3). Total RNA was extracted from 7.5% of cells pulsed with SILAC and AHA for each SILAC label and siRNA treatment. The integrity of the RNA was assessed using 2100Bioanalyzer (Agilent Genomics). Barcoded stranded RNAseq libraries were prepared from high quality total RNA samples (~200 ng/sample) using the Illumina TruSeq RNA Sample Preparation v2 Kit ??(Illumina, San Diego, CA, USA) implemented on the liquid handling robot Beckman FXP2. Obtained libraries that passed the QC step were pooled in equimolar amounts; 1.8 pM solution of this pool was loaded on the Illumina sequencer NextSeq 500 and sequenced uni-directionally, generating ~500 million reads, each 85 bases long.