title: NC2-L (CRX1025491; CRA016024)
library_construction_protocol: Briefly, 500 ng of each sample was fragmented using RNase III (Ambion) in a 20 microl mixture for 10 minutes at 37degreeC. The fragmented RNA was then purified using 40 microl of MagicPure RNA Beads (TransGen). Each RNA sample was subsequently divided into two halves, with one half used for proximity ligation and crosslink reversal (C, V samples). The proximity ligation process was then carried out using the following conditions: 200 ng of fragmented RNA, 1 unit/microl RNA ligase 1 (New England Biolabs), 1X RNA ligase buffer, 1mM ATP, 1 unit/microl Superase-in (Invitrogen), with a final volume of 200 microl. The reactions were incubated for 16 hours at 16degreeC and were stopped by cleaning with the miRNeasy kit (Qiagen). To reverse the crosslinking, the RNA was irradiated on ice with 254 nm UltraViolet C radiation for 5 minutes using a CL-1001 crosslinker (UVP). For the non-ligated controls, crosslink reversal was performed immediately after crosslinking, without proximity ligation (these controls were labeled as "-L").
identifiers.namespace: Beijing institute of biotechnology
identifiers.id: DRX716288
identifiers.namespace: NGDC
identifiers.id: CRX1025491