GSM2720018: uninfected BR2; Homo sapiens; OTHER
Protein G Dynabeads were coated with 2A8, a mouse monoclonal antibody against Argonaute (Ago).  Cells were lysed and treated with DNase and RNase T1.  The cell lysates were added to the antibody-coated beads and allowed to incubate overnight.  After immunoprecipitation, Ago-bound RNAs were phosphorylated.  Ago-RNA complexes were incubated overnight with RNA ligase.  This was followed by dephosphorylation and ligation of the miRCat-33 3'-linker (5'-TGGAATTCTCGGGTGCCAAGG-3').  After the complexes were eluted from the beads, the samples were treated with proteinase K.  The RNA was phosphorylated and ligated to the Illumina TruSeq® Small RNA Sample Prep Kits RA5 5' adapter. RNA was reverse-transcribed using Illumina TruSeq® Small RNA Sample Prep Kits RTP and then amplified through 19-24 PCR cycles with Illumina TruSeq® Small RNA Sample Prep Kits RP1 and one of the following barcoded primers: RPI1, RPI2, or RPI3.  WT_BR1, UI_BR1, and D11_BR1 received RPI1, WT_BR2, UI_BR2, and D11_BR2 received RPI2, and WT_BR3, UI_BR3, and D11_BR3 received RPI3.  PCR products were incubated with PEG-8000 and DNA was extracted from the supernatants.