GSM6215463
Argonaut High-throughput Sequencing UV-crosslinking and Immunoprecipitation (AGO-HITS-CLIP). Cell pellets were then subject to UV-crosslinking (25 kilograys), spun down, washed, and flash-frozen in liquid nitrogen, and stored at -80C. We then performed AGO-HITS-CLIP as described previously77,78 except for the use of photoactivatable ribonucleoside (PAR) analog thiouridine, as done in PAR-CLIP, which we did not include. Cell pellets were lysed (recipe ), DNAse (cat.) and RNAse (cat.) treated, and subject to immunoprecipitation of argonaut 2 (cat . ). Several high-salt stringent washes purified RISC-associated RNAs, proteinase (cat. ) treated, then 32P-radiolabeled, and size-selected by gel purification. These RNAs were then eluted and prepared for NGS using the TruSeq small RNA library (cat.) by ligations of the 3' and 5' adapters, reverse transcribed (cat.), and barcodes were added during PCR amplification of cDNA by the minimum number of PCR cycles (10-15 cycles) using a high-fidelity polymerase (cat.). TapeStation confirmed the purity of NGS libraries, and the libraries were single-end sequenced using the Illumina HiSeq 2500 platform (50-51 bp reads). Truseq small RNA