4 protocols
normalization data transformation protocol
Primary analysis performed using RTA followed by secondary analysis with CASAVA 1.8.1 Sequence analysis was conducted using CLC Genomics Workbench Version 4.9; reads were mapped to hg18 human genome build Unique exon reads for each gene were then used to create expression values for each gene with the following formula: Expression Value = (unique exon reads)(10^9)/(exon length)(total unique exon reads) A table was constructed consisting of genes with the respective associated expression values for each condition. Genome_build: hg18 Supplementary_files_format_and_content: text file contains genes and associated enrichment values for each sample. Fold change (Column 4) is the ratio of the respective values BJAB_3628/BJAB_3626 (Column_3/Column_2)
growth protocol
BJAB cells were grown in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (R10F) and 200 U/mL penicillin and 200 µg/mL streptomycin at 37°C in a 5% CO2 humidified atmosphere.
sample treatment protocol
Cell lines grown under the same conditions
nucleic acid library construction protocol
1×10^8 cells were washed with PBS and lysed with 500 µl of PLB buffer (10 mM HEPES, pH 7.0, 100 mM KCl, 0.5% NP-40, 5 mM MgCl2, 200 U/ml RNase inhibitor (Ambion), 1 mM DTT, proteinase inhibitor cocktail) at 4°C for 30 min. Following centrifugation, the lysate was incubated with anti-human Ago2 (11A9) antibody (Provided by Dr. Gunter Meister [Rüdel S, et al. (2008) A multifunctional human Argonaute2-specific monoclonal antibody. RNA 6: 1244–2153.]) conjugated dynabeads (Invitrogen) in 500 µl of NET-2 buffer (20 mM EDTA, pH 8.0, 1 mM DTT, and 200 U/ml RNase inhibitor in NT-2 buffer (150 mM Tris-HCl, pH 7.0, 100 mM Tris-HCl, pH 8.0, 750 mM NaCl, 0.25% NP-40, 5 mM MgCl2)) at 4°C for overnight. The protein-dynabead complexes were washed 6 times with NT-2 buffer, once with NTmS buffer (150 mM Tris-HCl, pH 7.0, 100 mM Tris-HCl, pH 8.0, 0.25% NP-40, 5 mM MgCl2) and resuspended with 1× Turbo DNase buffer and 2 U of Turbo DNase (Ambion) at 37°C for 30 min. The activity of DNase was eliminated with 15 mM of EDTA followed by addition of same volume of proteinase K buffer (2.4 mg/ml of proteinase K and 2% SDS in NT-2 buffer) at 55°C for 30 min. The RNA was extracted with acid-phenol/chloroform (pH 4.5) once, chloroform once and precipitated and resuspended in RNAse-free water. Libraries were generated using Illumina’s mRNA Seq kit (lot#5701543) Rev. D according to manufacturer's protocol. Briefly, samples were fragmented then precipitated at -80°C, followed by first strand & second strand cDNA synthesis. After end repair and adenylation of 3' ends, adapters were ligated to cDNA molecules. Following ligation, cDNA was purified using E-gel Size Select 2% agarose gels. Enrichment of purified cDNA was done via LMPCR (15 cycles) followed by purification using ZymoResearch DNA Clean & Concentrator columns (25ul elution volume). Libraries were validated on Agilent 1000 DNA chip and Quant-iT quantification.