shRNA vectors were purchased from Origene (Msk1: TR504795; MLL1: TR517798) and transfected into MEFs by electroporation (GenePulser XCell, Biorad). MEFs were brought into a single cell suspension by trypsination and washed with ice-cold PBS. 4x106 cells per transfection were resuspended in 400μl electroporation buffer (10mM Hepes, pH7.5, 135mM KCl, 2mM MgCl2, 5mM EGTA, 25% FBS) and transferred to a 4mm electroporation cuvette (Biorad). 20μg of vector DNA was added to the cells immediately before transfection and incubated on ice for 1 minute. Cells were transfected with one pulse (300V, 600μF, 1000Ω), and allowed to recover (RT, 1min), before addition of 10ml pre-warmed MEF medium and incubation at 37°C, 5% CO2 for 6 hours. Non-transfected cells were removed by puromycin treatment (10μg/ml, 14 hrs). After 20 hours cells were harvested.
Growth protocol
Mouse embryonic fibroblasts (MEFs) were generated in house, via isolation from Balb/c wild-type mouse embryos between day 12-15 of gestation and cultured in DMEM, 100 µg/ml streptomycin, 100 U/ml penicillin, 1/100 MEM non-essential amino acids, 1/1000 2-mercaptoethanol and 10% FBS at 37oC, 5% CO2.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using the RNeasy Mini kit (Qiagen) and DNA was removed by on-column DNase digestion with the RNase-Free DNase set (Qiagen). RNA was converted to double-stranded cDNA using cDNA synthesis kit (Roche Nimblegen, 11 117 831 001)
Label
cy3
Label protocol
Samples were labelled with Cy3 using a Nimblegen One Colour Labeling Kit (Roche Nimblegen, 06 370 438 001)
Hybridization protocol
Samples were hybridised using a Nimblegen Hybridisation Kit (05 583 934 001), including Sample Tracking Controls (05 223 512 001)
Scan protocol
Slides were scanned on a Nimblegen MS200 microarray scanner at a resolution of 2µm with balanced Speed/Sensitivity and 100% laser intensity using autogain
Description
Gene expression data following knockdown of KMT2A or MSK1 in mouse embryonic fibroblasts
Data processing
Data were extracted using DEVA (Roche Nimblegen) and normalised by robust multichip average in R.
