2100 bioanalyzer chip

RNA-seq analysis was performed by Capital Bio Technology (Beijing, China). Before preparation of the RNA-seq libraries, total RNA (1 μg) was treated with a RiboMinus Eukaryote Kit (Qiagen, Valencia, CA) to remove rRNA. Strand-specific RNA-seq libraries were prepared using a NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, Beverly, MA) following the manufacturer’s instructions. The libraries were quality controlled with a 2100 Bioanalyzer chip (Agilent, Santa Clara, CA) and sequenced on the HiSeq 2000 platform (Illumina, San Diego, CA) with 100 bp paired-end reads.
RNA sequencing reads were mapped to the GRCh38.p7 assembly using STAR aligner version 2.5.2b with Ensembl annotations. Data analysis was performed with R software with a low-stringency filtering scheme of a 1.5-fold change in the expression level after MAS5 normalization of all datasets. To investigate the global effect of circRNF220 on AML cells, we used KOBAS 3.0 (http://kobas.cbi.pku.edu.cn/index.php) for pathway analysis of the gene expression profile data for AML cells transfected with siRNA-circRNF220. Finally, Gene Set Enrichment Analysis (GSEA) v.2.0 was used to analyze a preranked list based on the DESeq2 t-statistic using preassembled gene sets from MSigDB v5.2.

Small RNA Library was prepared using TrueSeq small RNA library prep kit (Illumina San Diego CA, USA) according to the manufacturer's instruction. This whole procedure requires adapter ligation, reverse transcription, PCR amplification, and pooled gel purification to generate a library product. For targeting miRNAs having a 3′ hydroxyl group resulting from enzymatic cleavage by Dicer or other RNA processing enzymes, RNA 3′ adapter was specifically modified to target miRNAs. Further, Adapters were ligated to each end of the RNAs and subsequently reverse transcribed to create single-stranded cDNA and sequenced using miRNA sequencing on an IlluminaHiSeq 2000 platform (Illumina, San Diego, CA, USA). By using a DNA specific chip 1 μl of the resuspended library construct was loaded on Agilent Technologies 2100 Bioanalyzer chip for small RNA Library validation. Prior to the sequencing, all the individual libraries were clustered together using TrueSeq Cluster kit V3-cBot-HS (HiSeq) in a single lane on an IlluminaHiSeq 2000 platform that generated 0–100 bp paired end reads.