Gene expression profiling of SKOV-3 cells with or without PDK1 knocked down (Control versus siPDK1)

Created on 24 Aug 2019 - 01:28 by Michelle SiuTo examine the downstream targets of PDK1 in ovarian cancer. siRNA specifically targeting PDK1 (sc-36203) and control siRNA (Santa Cruz Biotechnology) were introduced into SKOV-3 cells using SilentFect for 48 hr. RNA was harvested using Trizol reagent. RNA preparation, library construction and sequencing with the BGISEQ-500 platform were performed at Beijing Genomics Institute (BGI, Shenzhen, China). 1) There are two methods to treat total RNA. Oligo (dT) magnetic beads are used to select mRNA with polyA tail, or hybridize the rRNA with DNA probe and digest the DNA/RNA hybrid strand, followed by DNase I reaction to remove DNA probe. Then obtain the target RNA after purification. 2) Fragment the target RNA and reverse transcription to double- strand cDNA (dscDNA) by N6 random primer. 3) End repair the dscDNA with phosphate at 5' end and stickiness 'A' at 3' end, then ligate and adaptor with stickiness 'T' at 3' end to the dscDNA. 4) Two specific primers are used to amplify the ligation product. 5) Denature the PCR product by heat and the single strand DNA is cyclized by splint oligo and DNA ligase. 6) Perform sequencing on prepared library. Gene expression levels were quantified using RNA-Seq by Expectation Maximization (RSEM) (Li and Dewey, 2011). The DEseq2 method was applied to detect differentially expressed genes (DEG) between groups. genome build: hg19Sample 1 (SK_C1_1): Control 1, SKOV-3
Sample 2 (SK_C2_1): Control 2, SKOV-3
Sample 3 (SK_siPD1_1): siPDK1 1, SKOV-3
Sample 4 (SK_siPD2_1): siPDK1 2, SKOV-3
SK_C-VS-SK_siPD.DEseq2_Method_GEO