Thymocytes or splenocytes from Cre-ERT2; Bcl11bflox/flox mice were cultured in T cell medium with 1 μM 4-OHT (Sigma) at 370 C for 48 hrs. After this time, cells were washed and resuspended with fresh media. T cell media: RPMI-1640, 10% FCS, 1% penicillin/streptomycin, 2 mM L-glutamine, 5 ng/ml muFlt-3-L, 5 ng/ml huIL-7.
Growth protocol
After 4-hydroxytamoxifen (OHT) treatment, DN3 thymocytes from Bcl11bCKO/CKO and Bcl11bCKO/+ were sorted by FACS and co-cultured with OP9-DL1 in T cell culture media (3,000 cells per well in 24-well plates) with 100 ng/ml huIL-2, respectively. Every three days, cells were collected by vigorous pipetting, filtered through cell strainers and transferred to new tissue culture plates pre-seeded with fresh OP9-DL1 stromal cells. Fourteen days after OHT treatment, cells were collected for RNA extraction. For ex vivo expansion, splenic ITNK cells were enriched using the NK Isolation Kit (Miltenyi) and cultured for 6-9 days at 1 x 106 cells/ml in RPMI 1640 medium containing 10% FCS/50 µM 2-mercaptoethanol/2.0 mM L-glutamine and 1000 U/ml hIL-2 (Chiron).
Extracted molecule
total RNA
Extraction protocol
RNA from primary cells was extracted using the RNAqueous-Micro RNA isolation kit according to the manufacture protocol (Ambion). Briefly, cells were counted and pelleted by centrifuge at 4,000 G for 5 min. Remove supernatant thoroughly by aspiration. Resuspend cell pellet by vortexing vigorously in at least 100 ul Lysis Solution. Then 50 ul of 100% ethanol was added and mixed with lysate thoroughly. Next load the lysate/ethanol mixture onto a Micro Filter Cartridge Assembly and centrifuge for 10 sec at 16,000 G. Then wash the filter with 180 ul of Wash Solution 1 once and 180 ul of Wash Solution 2/3 twice. Discard the flow-through and centrifuge the filter for 1 min at 16,000 G. Elute the RNA into a Micro Elution Tube with 2X5 ul preheated Elution Solution. To eliminate contaminated DNA from extracted RNA, 1/10th volume of 10X DNase I Buffer and 1 ul of DNase I were added into the RNA sample and mixed gently but thoroughly. After incubation at 20 min at 370C, the DNase reaction was mixed with 2 ul DNase Inactivation Reagent and left at room temperature for 2 min. Finally, pellet the DNase Inactivation Reagent and transfer the RNA to a fresh tube. For RT-PCR or qRT-PCR experiments, RNA quality and quantity was verified using a Nanodrop ND-100 Spectrophotometer (Thermo Scientific). For gene expression array experiments, RNA quality and quantity was determined using 2100 Bioanalyzer platform (Aglient Technologies).
Label
biotin
Label protocol
Biotinylated cRNA were prepared with the Ambion MessageAmp kit for Illumina arrays
Hybridization protocol
Standard Illumina hybridization protocol
Scan protocol
Standard Illumina scanning protocol
Description
replicate 1
Data processing
The data were normalized using quantile normalization with dChip
