title: GSM8977054: Hfq, K31A-FLAG, input, 2; Escherichia coli; OTHER
library_construction_protocol: The proteins and RNA molecules were crosslinked in vivo by exposing the cells to 800 mJ of 254 nm UV irradiation. After lysing the cells mechanically, Hfq and its bound RNAs were coimmunoprecipitated using either anti-Flag antibody or Hfq antibody. RNAs were trimmed by RNase A/T1 and then treated by T4 polynucleotide kinase (PNK) to generate fragments with 5' P and 3' OH ends that can be subsequently ligated. T4 RNA ligase was then used to ligase the neighboring RNAs. After digesting Hfq protein by proteinase K, the RNAs were isolated and subjected to sequencing library construction. cDNA libraries were constructed based on a protocol adapted to capture the bacterial sRNAs as well as mRNAs (Melamed et al., 2018, Nat. Protoc., 13, 1-33). 400 ng of total RNA samples or Hfq co-IP RNA samples were used as input. 12 cycles of PCR were performed at the last step to enrich each library.
identifiers.namespace: GEO
identifiers.id: GSM8977054_r1