Rneasy kit with dnase treatment

MDA231 cells were treated with (i) vehicle, (ii) 5 μM MK-4827 (PARPi), (iii) 2.5 μM AZD-1775 (Wee1i) and (iv) combination of MK-4827 and AZD-1775 for 48 h and cultured in drug free media for an additional 48 h, harvested and total RNA from each sample was isolated using a RNeasy Kit with DNase treatment (#74104 and #79254, Qiagen, Germantown, MD, USA). RNA samples were submitted to the Sequencing and Microarray Core Facility at MD Anderson Cancer Center. mRNAs were enriched by Poly(A) selection, and the libraries were prepared by mRNA-Seq Sample Prep Kit (Illumina, Foster City, CA, USA) following the manufacturer’s instructions. Pair-end 76-bp sequencing was performed by Illumina HiSeq 2000. FASTQ sequence files were obtained, and the RNA-seq reads were aligned to the human reference genome. Functional analysis of the differentially expressed transcripts was performed using gene set enrichment analysis (GSEA) as described [73 (link)].

RNeasy kit with DNase treatment (Qiagen, Germantown, MD, USA) was used to isolate RNA from flash frozen cell pellets. The iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) was used to convert 1 µg of RNA to cDNA. Quantitative reverse PCR (qPCR) was performed with iTaq universal SYBR green supermix (Bio-Rad, Hercules, CA, USA) in the C1000 Touch Thermal cycler (Bio-Rad, Hercules, CA, USA). Data was analyzed using CFX manager 3.1 (BioRad, Hercules, CA, USA). Levels of GAPDH expression were used for normalization. Statistics were determined by Student’s unpaired t-test. The forward and the reverse primers used for the qPCR reactions are as follows: PSMB7 (5′-TGC AAA GAG GGG ATA CAA GC-3′ and 5′-GCA ACA ACC ATC CCT TCA GT-3′), PSMD12 (5′-GTG CGC GAC TGA CTA AAA CA-3′ and 5′-TAG GCA GAG CCT CAT TTG CT-3′), and GAPDH (5′-AAC TTT GGC ATT GTG GAA GG-3′ and 5′-GGA TGC AGG GAT GAT GTT CT-3′).

RNeasy kit with DNase treatment (Qiagen, Germantown, MD, USA) was used to isolate RNA from frozen cell pellets. The iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) was used to convert 1 µg of RNA to cDNA. Quantitative reverse PCR (qPCR) was performed with iTaq universal SYBR green supermix (Bio-Rad, Hercules, CA, USA) in the C1000 Touch Thermal cycler (Bio-Rad, Hercules, CA, USA). Data was analyzed using CFX manager 3.1 (BioRad, Hercules, CA, USA). Levels of GAPDH expression were used for normalization. Statistics were determined by Student’s unpaired t-test. The forward and the reverse primers used for the qPCR reactions are as follows: PSMB4 (5′-TTC ACT GGC CAC TGG TTA TG-3′ and 5′-CGA ACG GGC ATC TCT GTA GT-3′), PSMB7 (5′-CTG TCT TGG AAG CGG ATT TC-3′ and 5′-GCA ACA ACC ATC CCT TCA GT-3′), PSMC4 (5′-TGG TCA TCG GTC AGT TCT TG-3′ and 5′-CGG TCG ATG GTA CTC AGG AT-3′), PSMD12 (5′-TCA CAG ACC TGC CAG TCA AG-3′ and 5′-AGG TTT TAG TCA GCC GAG CA-3′), and GAPDH (5′-AAC TTT GGC ATT GTG GAA GG-3′ and 5′-GGA TGC AGG GAT GAT GTT CT-3′).