GSM5015722: DroshaKO_BR1_R1; Homo sapiens; OTHER
Protein G dynabeads were conjugated with 2A8 anti-Ago while Protein L beads were conjugated with 4F9 anti-Ago. Pellets were resuspended in lysis buffer and incubated with RQ1 Dnase. Lysates were quantitated and added to conjugated beads overnight. Following immunoprecipitation, Ago bound RNAs were biefly treated with RNAseA, vigouraously washed, and phosphorylated before being subjected to high concentration of RNA ligase for intermolecular ligation. After overnight incubation, RNAs were dephosphorylated and the miRCat-33 linker was ligated. Samples were eluted from the beads, subjected to proteinase K digestion to release RNA complexes from Ago, and phosphorylated for ligation of the 5' adapter. RNA was reverse transcribed and the resulting cDNA was used for library prep. cDNA containing the miRCat-33 and 5-adapter adapter was PCR amplified using Illumina TruSeq® Small RNA Sample Prep Kits. The resulting library was seperated with Native-PAGE and DNA from 147bps and 527bps was extracted for Illumina sequencing.