A p53/LINC00324 positive feedback loop suppresses tumor growth by counteracting SET-mediated transcriptional repression
The cells were collected in TRIzol reagent, and total RNA was extracted according to the manufacturers instructions (Invitrogen). Genomic DNA was removed by treatment with DNase I (TaKara, Shiga, Japan). Then, RNA quality was determined by a 2100 Bioanalyzer (Agilent, CA, USA), and the amount of RNA was quantified using an ND-2000 (NanoDrop, Thermo Fisher Scientific, MA, USA). Only high-quality RNA samples (OD260/280=1.8~2.2, OD260/2302.0, RIN6.5, 28S:18S1.0, >1 g) were used to construct a sequencing library. RNA-sequence analysis was performed at Shanghai Majorbio Bio-Pharm Technology Co. Ltd, China. Briefly, an RNA-seq transcriptome library was prepared with the TruSeqTM RNA sample preparation kit from Illumina (San Diego, CA, USA) using 1 g of total RNA. The messenger RNA was isolated with oligo (dT) beads and then fragmented with fragmentation buffer. Double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen) with random hexamer primers (Illumina). Then, the synthesized cDNA was subjected to end repair, phosphorylation and A base addition according to Illuminas library construction protocol. Libraries were size selected to include cDNA target fragments of 200-300 bp with 2% Low Range Ultra Agarose followed by PCR amplification using Phusion DNA polymerase (NEB, MA, USA) for 15 PCR cycles. After quantification by TBS380 (Picogreen), the paired-end RNA-seq sequencing library was sequenced with the Illumina HiSeq xten/NovaSeq 6000 sequencer (2  150 bp read length). The raw paired-end reads were trimmed and quality controlled by SeqPrep and Sickle with default parameters. Then, the clean reads were separately aligned to the reference genome with orientation mode using HISAT2 software. The mapped reads of each sample were assembled by StringTie with a reference-based approach.