GSM5322361: EC_BW25113_CoIP_SpyCas9-3xF_native_leader_array_R2; Escherichia coli; RIP-Seq
Briefly, the cells harboring corresponding plasmids were lysed, tagged proteins were enriched through immunoprecipitation, and RNA bound to the tagged proteins were extracted using phenol-chloroform-isoamyl alcohol method for RIP-seq. The cells harboring the plasmids encoding untagged proteins were subjected to the same extraction procedures to use as controls. The extracted RNA was treated with DNase I (Thermo Scientific, EN0525) following the manufacturer's instructions. cDNA libraries for Illumina sequencing were constructed by Vertis Biotechnologie AG, Germany (http://www.vertis-biotech.com). Briefly, the resulting RNA was subjected to oligonucleotide adapter ligation on the 3′ end, first-strand cDNA synthesis using M-MLV reverse transcriptase (Agilent), and Illumina TruSeq sequencing adapter ligation on the 3′ end. The resulting cDNA was PCR-amplified using Herculase II Fusion DNA Polymerase (Agilent) with 13 amplification cycles following the manufacturer's instructions, purified using Agencourt AMPure XP kit (Beckman Coulter Genomics) following the manufacturer's instructions, and analyzed by capillary Electrophoresis.