cell line: NIH3T3 cell type: fibroblasts infected with: MCMV ip antibody: anti-Ago2 mAB (6F4) ip antibody provider: a kind gift of G. Meister, published in Zhu et al. J Virol 84, p. 10266 5'-barcode: CTG
Infection with MCMV at MOI of 10, 72 hrs. For immunoprecipitation, extracts were prepared from cells after 72 h of infection and the proteins were IP'ed with anti-Ago1 (Wako mAB 2A7, cat. no. 015-22411, lot STM2295) or anti-Ago2 (mAB 6F4, a kind gift of G. Meister, antibody published in Zhu et al. J Virol 84, p. 10266 ff), followed by RNA extraction with Trizol.
cells were grown in DMEM with 10 % FCS
Extraction of total RNA with Trizol. Gel purification of small RNAs (17-30nt). 3'- (AMP-5’p=5’pCTGTAGGCACCATCAATdideoxyC-3’) and 5'- (5'-rArCrArCrUrCrUrUrUrCrCrCrUrArCrArCrGrArCrGrC rUrCrUrUrCrCrGrArUrCrU-3' + 3 nt Barcode (-CUG, -GAU, -CCU)) Adapter ligation, reverse transcription + PCR to append Illumina bridge amplification sequences
Illumina Genome Analyzer IIx
total RNA after IP, size-selected (17-30 nt)
The sequences in the fastq file were sorted according to the 5'-Barcodes and the barcode was removed. (resulting file: *_no_barcode.fastq, ad-hoc PERL script, available upon request) The 3'-linker was removed (ad-hoc PERL script, available upon request) The remaining small RNA sequences were filtered for length between 17 and 23 nucleotides (ad-hoc PERL script, available on request) sample data table: Sequences were sorted and reduced to a unique set with the LINUX shell commands sort and uniq -c