title: GSM1552212: Gene expression miR-191 BioRep1_seq; Homo sapiens; RNA-Seq
library_construction_protocol: 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.