GSM1893159: cKO ctrl-a; Mus musculus; RNA-Seq
Animals were sacrificed by cervical dislocation and hippocampi were removed bilaterally and subjected to total RNA extraction by TRIzol (Invitrogen #15596-018) as per manufacturer instructions. Polyadenylated RNA was purified by Dynabeads® mRNA purification kit (Life Technologies #61006). As per manufacturer protocol, 1µg of total RNA was incubated at 50°C for 2 min and placed on ice. The sample was added to Dynabeads® with 50µL binding buffer, mixed thoroughly at room temperature for 5 min, washed twice, and then eluted into 10 µL 10 mM Tris-HCl (pH 7.5) by heating to 70°C for 2 min. Purified mRNA was treated with DNase I (Life Technologies #18068015) at 37°C for 30 min followed by 70°C for 5 min. For first strand synthesis, 10µL of the digestion product, 1µL oligo(dT)18 (500 ug/ml), and 1μl 10 mM dNTP mix were mixed. Samples were heated to 65°C for 5 minutes and incubated on ice. Four microliters of 5X First-Strand Buffer, 2μl of 0.1 M DTT, and 1μl of RNaseOUT Recombinant RNase Inhibitor (Life Technologies #10777-019) were added and incubated at 37°C for 2 min. One microliter of MMLV reverse transcriptase (Life Technologies #26025) was added to samples, which were then incubated at 37°C for 50 minutes, followed by a five-minute inactivation at  70°. cDNA was then treated with NEBNEXT mRNA second strand synthesis  mix (neb #e6112) and incubated for 2.5 hours at  16°C, followed by sonication for 10 seconds.  Samples were purified on a PCR column (Fermentas #K0702) and eluted in 22µl 10 mM Tris pH 8. Blunt ends were prepared using the DNATerminator® End Repair Kit (Lucigen #40035). Samples were purified using AMPure xp beads (Beckman #A63880) and A-tailing of 3’ ends of eluted cDNA was performed using Exo-minus Klenow DNA polymerase (Epicentre #KL0810259) at 37°C for 30 min. Uniquely tagged Illumina genomic adopters were ligated by Enzymatics T4 DNA ligase (Invitrogen #15224) at RT for 2hrs.  Size visualization was performed using a 2% agarose gel, yielding cDNA libraries ranging in size from 200–250 bp, which were amplified by PCR (5 sec at 98°C; 10 sec at 72°C; 10-20 cycles) using Phusion High-Fidelity DNA Polymerase (ThermoScientific #F530) .