|Public on Mar 01, 2015
|Gene expression miR-191 BioRep3_seq
|cell line: HeLa
|mature miR-191 duplex or control siRNA against GFP duplex was transfected with lipofectamine 2000 at a concentration of 100nM in the well
|DMEM supplemented with 10% FBS
|Lysates from each sample were split into 2 groups. Group 1: Gene expression samples, total RNA was isolated with TRIzol. Gene 2: Ago2 immunoprecipitation samples, Ago2 was immunoprecipated with an antibody against Ago2 and the associated RNA was isolated with TRIzol. In more detail, 24 hours post transfection, cells were washed 2X with PBS, 0.5 mL of lysis buffer was added to the plate, followed by incubation at 4°C for 30 minutes. Cell lysates were collected by scraping and debris cleared by centrifugation at 14,000 rpm at 4°C. 50 μl of the lysate was collected as input for total RNA profiling. Lysates were pre-cleared by incubating with 50 μl of protein-G beads (Roche) for 1 hour at 4°C and then collecting supernatants (pre-clearing). Pre-cleared lysates were then incubated with 15 μg of antibody against Argonaute-2 (AGO2)(ab57113, Abcam) at 4°C for 3 hours. AGO2 is a protein component of RISC. Following AGO2 antibody incubation, 50 μl of protein-G beads were added to the lysate and incubated for 1 hour at 4°C. Beads were washed 8 times with lysis buffer and RISC-RNA complexes were extracted by adding 1 mL TRIzol reagent (Invitrogen) directly to the beads. RNA extraction with TRIzol was carried out as per the manufacturers instructions.
rRNAs were removed using the Ribo-Zero rRNA Removal Kit (Epicentre). The remaining RNA was then fragmented with the NEBNext Magnesium RNA Fragmentation Module (New England BioLabs), and size selected using AMPure XP beads (Agencourt) at 1.8X volume. Ends of the fragmented RNAs were then prepared for adaptor ligation using T4 Polynucleotide Kinase (New England BioLabs). Libraries were prepared from the RNA using the NEBNext Small RNA Library Prep Set according to the manufacturers protocol (New England BioLabs). Libraries were cleaned and further size selected using 0.8X volume AMPure XP beads, and additional size selections with 1.0X beads were performed as necessary.
|Illumina HiSeq 2500
|Illumina CASAVA version 1.8 used for basecalling.
Cutadapt was used to trim adaptors, reads mapping to rRNAs and tRNAs were removed, and the remaining reads were mapped to human genome (hg19) by TopHat 2 software version 2.0.9 with parameters -p 6 --b2-fast --library-type=fr-unstranded --no-novel-juncs --no-novel-indels --transcriptome-index
Cuffdiff was used to combine biological replicates and assign expression values (FPKM) for each RefSeq gene, run with parameters -p 1 -b -u
Supplementary_files_format_and_content: tab-delimited text files include FPKM values (value 1 and value 2) from cuffdiff combined biological replicates 1-3 for each treatment group and significance estimates comparing miR-191 transfection to control transfection
|Nov 21, 2014
|Last update date
|May 15, 2019
|University of Texas at Austin
|2500 Speedway Dr. MBB 3.212
|miR-191 regulates human cell proliferation and directly targets multiple oncogenes [seq]
|Genome wide miR-191 target profile determined by RIP and gene expression profiling