title: GSM8208399: adult forebrain, cell-selective, rep3; Mus musculus; OTHER
library_construction_protocol: Mouse brain tissue in Homogenization buffer (1X HBSS supplemented with 5 mM MgCl2, 10 mM HEPES (pH 7.4) and 1X HALT protease/phosphatase inhibitor) is homogenized by 12 passes through a 21G needle. UV irradiate homogenates 3x with (4000 µJ/cm2 x 100) on a perfluoroalkoxy (PFA) petri dish, mixing samples between rounds. Transfer irradiated tissue homogenate to a microfuge tube and centrifuge 900 x g for 5 minutes at 4 °C.  Immunoprecipitation from the cell pellets were done with anti-Ago2 (2D4, Wako, 018-22021)  for total CIMERA-seq, anti-myc (9B11, Cell Signaling, 2276) for cell-selective CIMERA-seq, and anti-IgG1 (Invitrogen, 14-4714-82) for negative control. Libraries were prepared by intermolecular ligation of small non-coding RNA and target RNA, RNA isolation from Ago protein, ligation of 3' and 5' adaptors, reverse transcription, minimal PCR amplification, gel purification and size selection. custom sequencing libraries with 150 base-pair paired-end sequencing and a read depth of 61 – 138 million reads per sample (Illumina HiSeq X or NovaSeq)