GSM6576142
Genome editing using CRISPR-U was used to knockout the lncRNAs' DNA binding domains (DBDs). Before and after the knockout, RNA-sequencing was performed to examine gene expression (performed by Novogene, Beijing, China, http://cn.novogene.com). Ribo-Zero Magnetic Kit is used to remove rRNA before constructing the RNA-seq libraries. By using the fragmentation buffer, the mRNAs and non-coding RNAs are fragmented into short fragments(about 200~500nt), then the first-strand cDNA is synthesized by random hexamer-primer using the fragments as templates, and dTTP is substituted by dUTP during the synthesis of the second strand. Short fragments are purified and resolved with EB buffer for end reparation and single nucleotide A (adenine) addition. After that, the short fragments are connected with adapters, then the second strand is degraded using UNG(Uracil-N-Glycosylase) finally. After agarose gel electrophoresis, the suitable fragments are selected for the PCR amplification as templates. During the QC steps, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System are used in quantification and qualification of the sample library. RNA libraries were prepared for sequencing using standard Illumina protocols.