title: Illumina HiSeq 2000 sequencing; Gene expression profiles of H16N2 cells expressing EZH2 variants
design: Gene expression profiles of H16N2 cells expressing EZH2 variants
library_construction_protocol: H16N2 cells were cultured at 37 degree celsius with 5% carbon dioxide  in F12 Medium supplemented with 5 ug/ml Insulin-transferrin-Sodium selenite, 1 mg/ml Hydrocortisone, 10 ng/mL EGF, 25 ug/ml Gentamicin, 2.5 ug/ml plasmocin, 0.3 ul/ml Ethanoamine, 10mM HEPES, 1 mg/ml Bovine Serum Albumin. 2% of Fetal Calf Serum was supplemented for 24 hours after re-platting. Cells were detached from the culture plates by treatment with 0.05% Trypsin/EDTA at 37 degree celsius for 3 minutes H16N2 cells were first transduced with empty vector or MIG retroviral vector carrying different EZH2 variants. After recovery from transduction, cells successfully transduced by MIG retroviral vector were sorted in flowcytometer based on GFP reporter. GFP positive cells were transduced a second time with shRNA targetting endogenous/human EZH2 in lentiviral vector. 10 ug/ml of puromycin was used to select for H16N2 cells that were transduced by the lentiviral vector. For expression analysis, total RNA from the cells was extract using QIAshredder (Qiagen) and RNeasy kit (Qiagen) according to manufacturer's protocol.  Briefly, cells lysed in guanidine salt-containing buffer were centrifuged in QIAshredder for homogenization. RNA was precipitated by addition of  1 volume of 70% ethanol and captured in RNeasy column. RNeasy column with bound RNA was washed 3 times with buffers provided and eluted in RNase-free water. No DNase I treatment was performed as the RNeasy columns do not bind genomic DNA. Library Preparation (Stranded mRNA-seq) The total RNA quality and RIN value was confirmed with a 2100 Bioanalyzer from Agilent using their RNA 6000 Nano chip. A high RIN value of 8 and above indicates that the total RNA quality was good and suitable for library preparation. Library preparation was done using a commercially available kit, Illumina® TruSeq Stranded mRNA LT Sample Prep Kit, following the manufacturer’s protocol. During the library construction, the enriched mRNA was fragmented to obtain inserts with fragment size of 120–200bp (a median size of 150 bp). The quality of the library was checked using both Agilent Bioanalyzer DNA1000 chip and KAPA quantification. A single peak in the expected region of ~280bp was observed indicating that the library was good and suitable for sequencing.
attributes.tag: Experimental Factor: compound
attributes.value: NA
attributes.tag: LIBRARY_STRAND
attributes.value: not applicable