alias: GSM3409377
title: GSM3409377: fertilized egg NAIN3; Danio rerio; OTHER
library_construction_protocol: The  RNA or modified RNA was extracted or cleaned up and eluted in RNase-free water by using TRIzol® Reagent (invitrogen). For icSHAPE, the in vivo modified RNA and unmodified RNA were biotinylated by copper-free click reaction, followed by fragmentation. The fragmented RNA was end repaired by incubation at 37°C for 1 hours with the following mix (70 mM Tris 7.0, 18mM MgCl2, 5mM DTT, 4 U/µl RiboLock, 0.1 U/µl FastAP (Life Technology), 2 U/µl T4 PNK(NEB)) before 3' adapter ligation with addition of 10 µl ligation mix (5mM DTT, 0.5µM 5' adenylated and 3'-blocked linker (3'-bio for unmodified, 3'-ddc for modified), 0.66 U/µl T4 RNA ligase (NEB, M0437M), 15% PEG8000, 1X RNA ligase buffer) and incubation at 25°C for extra 3 hours. Then the excess adaptor was removed as described below. The purified RNA was incubated in the mix of 1µl FastAP, 0.2µl SSB (Promega), 0.8µl Ribolock and 1µl 5' Deadenylase (NEB) in 1X NEB buffer 2 (NEB) at 30°C for 90 min. And then 1µl RecJf (NEB) was added with another incubation at 37°C for 1 hour. The following procedures, including reverse transcription, biotin-streptavadin enrichment, size selection of cDNA, circularization and PCR amplification, were the same as described in the standard protocol. For RNA-seq， Fragmented mRNA was used for library construction using the KAPA Stranded mRNA-Seq Kit (KAPA) according to manufacturer's protocol.
identifiers.namespace: GEO
identifiers.id: GSM3409377
attributes.tag: GEO Accession
attributes.value: GSM3409377