GSM6476940: HITS-CLIP Flag-Ago2b; Mus musculus; RIP-Seq
In three independent experiments (experiment 1-3), mice that express a Flag-tagged Ago2 protein specifically in Camk2+ neurons in the forebrain (Camk2a-cre; Rosa-Stopfl/fl-Flag_x0002_Ago2) and control mice (Rosa-Stopfl/fl-Flag-Ago2) were sacrificed at 8 to 14 weeks of age. Forebrains were excised, triturated to disrupt tissue, and UV-irradiated three times in a Stratalinker with 400mJ/cm2. Immunoprecipitation was performed as described (8) with the following modifications to the protocol: After UV crosslinking the frozen lysates were sonicated to improve tissue homogenization. RNase digestion with RNase A was performed before IP (using one fourth the concentration of RNase A used by (8)), and after IP (using one twentieth the concentration of RNase A used by (8)). 30µl of magnetic beads pre-coupled to the M2 FLAG antibody (M8823, Sigma) were used for IP on half of one forebrain after pre-blocking the beads with 2% BSA, 1% t-RNAs and 1% salmon sperm for 1.5 hrs at 4o C. RNA was directly de-crosslinked from protein after extensive washing The small RNA sample preparation kit, version 1.5 (Illumina), was used for library preparation with subtle modifications. Because low concentrations of RNA were purified from the CLIP procedure, as measured by Quant-iT RiboGreen RNA Assay (Invitrogen), the following modifications were made to the small RNA sample preparation kit (Illumina) to allow for low RNA input: Both the v1.5 sRNA 3' Adapter and the SRA 5' Adapter were diluted 10 times in nuclease-free water compared to the standard protocol before adapter ligation. The RT reaction was doubled (resulting in a final RT volume of 20 µl instead of the standard 10 µl), all of which was used for the downstream PCR reaction. To allow for the larger volume of RT product, 10 µl less water was used in the PCR step, resulting in a final PCR volume of 50 µl. Instead of the 12 cycles of PCR directed in the standard protocol 15 cycles were performed to increase output. RNA from four CLIP procedures were pooled for each library preparation, amounting to CLIP RNA from two mouse forebrains per library. At the end of the Illumina small RNA protocol, the products were purified from a gel at the size of 110 to 150 bp, containing adapter-linked RISC-associated mRNA target sites