GSM4721514: FH-Piwi-IP_3_3; Drosophila melanogaster; RIP-Seq
piRNAs: RNA was extracted from the OSC cell lysate by immunoprecipitating PIWI proteins using anti-Flag antibody beads or custom made fly anti-Piwi antibodies. Total small RNAs: RNA was extracted from the cells usign PureLink miRNA Isolation kit (Thermo Fisher Scientific, K157001) piRNA libraries: Imunoprecipitated RNA was ligated to 3` adapter containing unique indexes and two random bases (2N), and ligation products in 48 to 58-nt size range (equivalent to 19 - 29-nt long initial fragments) were recovered from Urea PAGE gel. Purified fragments were then ligated to 5` adapter containing eight random bases (8N) and purified on Urea PAGE gel again by selecting fragments in 82 - 92-nt size range. Final fragments were PCR amplified to add sequencing adapters. Total small RNAs libraries: A pellet of one million cells was used to purifiy small RNAs using PureLink miRNA Isolation kit (Thermo Fisher Scientific, K157001) and were spiked with a mix of 26 nt-long synthetic RNA callibrator sequences. RNA was ligated to 3` adapter containing unique indexes and two random bases (2N), and ligation products in 48 to 58-nt size range (equivalent to 19 - 29-nt long initial fragments) were recovered from Urea PAGE gel. Purified fragments were then ligated to 5` adapter containing eight random bases (8N) and PCR ampliffied to add sequencing adapters.
GEO Accession
GSM4721514