GSE175434
miR-378-3p Knockdown Mimics of MDS
The files represent mRNAseq data of the HL60 cell line (untransduced), and duplicate samples of HL60s transduced with empty (scrambled) control vector (System Biosciences, Cat. #MZIP000-VA-1) and duplicate samples of HL60s transduced with miRZip anti-miRNA Expression lentivector for miR-378-3p (System Biosciences, Cat# CS970A-1). Overall design: miRZip-378a-3p (ZIP) and scramble (SCR) pGreenPurTM lentivectors (Cat# CS970A-1, System Biosciences, Palo Alto, CA) encode either a miRZip hairpin interfering RNA designed to generate the full-length antisense miR-378a-3p or an empty vector, respectively. HL60 were seeded in 24-well plates and infected with a multiplicity of infection of 20 with ZIP or SCR using polybrene transduction reagent (8ug/mL) (Cat# TR-1003-G, Millipore, Burlington, MA) by spin transduction (1500g, 2 hours). Initial transduction efficiency was measured by flow cytometry on day 3. After puromycin (Cat# P8833; Sigma Aldrich; Burlington, MA) (2ug/mL) treatment on day 3 through day 8, selection efficiency was measured by flow cytometry.  Expression of the ZIP construct normalized to U6 was confirmed by real time polymerase chain reaction (qPCR) using QuantiMirTM (Cat#RA420A-hU6, System Biosciences), using the ABI 7300 Real Time PCR System (Applied Biosystem, Foster City, CA).  Expression of miR-378-3p normalized to U6 was assessed by qPCR using TaqManTM MicroRNA Assays (Cat# 4427975, Applied Biosystems). Presence of green fluorescent protein (GFP) in both ZIP and SCR vectors was confirmed by polymerase chain reaction (PCR) and fluorescent microscopy (Nikon Eclipse TE2000-S Inverted microscope, Melville, NY). Library construction was conducted on total RNA using the TruSeq Small RNA sample preparation kit (Illumina, San Diego, CA) and sequenced on an Illumina HiSeq 2500. The raw sequencing reads in BCL format were processed through CASAVA-1.8.2 for FASTQ conversion and demultiplexing.  The RTA chastity filter was used, and only the pass filter reads were retained for further analysis. RNA-seq data went through multi-perspective quality control procedures following established guidelines. Alignment was performed by TopHat 2. Gene quantification using both Cufflinks and HTSeq was converted to differential expression analysis using Cuffdiff from the Cufflinks package and MultiRankSeq, respectively, which is a combination of DEGseq, edgeR, and baySeq.  A list of the most differentially expressed genes was generated from all four methods based on a weighted flexible compound covariate method (WFCCM).