alias: GSM3304660
title: GSM3304660: dictionary sequencing; synthetic construct; OTHER
library_construction_protocol: pri-miRNA variants containing 29–31-nucleotide barcodes and random sequences at two junctional positions were prepared by oligonucleotide annealing, extension, and PAGE gel purification. After quantification of the initial pools using NEBNext library quantitation kit (NEB), 16,000,000 molecules per pri-miRNA group were reamplified by PCR to decrease barcode complexity. An aliquot from the amplified DNA was sequenced as "dictionary". Next, T7 in vitro transcription was used to produce pri-miRNAs from the above DNA pools, and a subset of the RNA was sequentially treated with alkaline phosphatase and T4 polynucleotide kinase to be traced using gamma ATP. An aliquot from the transcribed RNA was stored as "input". In vitro DROSHA processing was carried out to the "input" pri-miRNAs, and 5′ cleavage fragments were purified and ligated with a 3′ adapter which contains 4-nucleotide random sequences. Next, reverse transcription using SuperScript III (Thermo Fisher) was carried out on "cleavage fragment" as well as "input" RNAs. After amplification using PCR, a 6% native PAGE gel was used to purify DNA libraries. Illumina adapter sequences were incorporated to the libraries using PCR.
identifiers.namespace: GEO
identifiers.id: GSM3304660