title: GSM5961623: YhgF minus crosslink biorep 2; Escherichia coli str. K-12 substr. MG1655; RIP-Seq
library_construction_protocol: The YhgF protein was purified using anti-FLAG beads and bound RNA was partly digested and labeled with P32. The samples were separated on polyacrylamide gels andtransferred to nitrocellulose membranes. The RNA was prepared from the membrane by cutting out the pieces cotaining protein. Co-purifying RNA was identified by deep sequencing Libraries were prepared using the NEBNext multiplex small RNA library prep set for Illumina (New England Biolabs) according to the manufacturer's directions, except that 20 cycles of PCR was done after the reverse transcription. MinElute PCR purification kit (Qiagen) was used to purify and up-concentrate PCR samples. The amplified libraries were size separated on a 6 % polyacrylamide gel, stained with SYBR gold (Thermo Fisher) and fragments in the size of 140-250 bp excised from the gel. The DNA was purified using the elution buffer supplied with the kit and overnight incubation at 16 °C while shaking at 1200 rpm. To remove gel fragments from the samples, they were centrifuged through a Costar Spin-X column (0.45 µl cellulose membrane, Corning) and ethanol precipitated using linear acrylamide (from kit) as coprecipitant. To amplify the DNA library, another nine rounds of PCR was done followed again by purification and up-concentration using MinElute PCR purification kit