title: GSM8036246: Cerebellum, input, WT, rep3; Mus musculus; RNA-Seq
library_construction_protocol: TRAP was performed as described in (Doyle et al. 2008 and Heiman et al. 2008). Briefly, Streptavidin MyOne T1 Dynabeads were coated with biotinylated protein L for 35 min at room temperature, washed with 3% IgG and Protease-free BSA in 1× PBS and subsequently incubated with 50 µg each of 19C8 and 19F7 anti-GFP monoclonal antibodies (Memorial Sloan-Kettering Monoclonal Antibody Facility) in 0.15 M KCl TRAP wash buffer (10 mM HEPES-KOH at pH 7.4, 5 mM MgCl2, 150 mM KCl, 1% NP-40, supplemented with 100 µg/mL cycloheximide in methanol, 0.5 mM DTT, 20 U/mL RNasin) for 30 min at room temperature using end-over-end rotation. After antibody binding, the beads were washed three times with 0.15 M KCl TRAP wash buffer and resuspended in 0.15 M KCl TRAP wash buffer. Each reaction was supplemented with 30 mM DHPC. Cerebella from P21 Tg(Pcp2-Egfp-L10a) mice were dissected and placed in TRAP dissection buffer (2.5 mM HEPES-KOH at pH 7.4, 35 mM glucose, 4 mM NaHCO3 in 1× HBSS, supplemented with 100 µg/mL cycloheximide). The tissue was homogenized in chilled TRAP lysis buffer (10 mM HEPES-KOH at pH 7.4, 5 mM MgCl2, 150 mM KCl, supplemented with 0.5 mM DTT, 100 µg/mL cycloheximide, protease inhibitor cocktail, 40 U/mL RNasin, and 20 U/mL Superasin). The homogenate was centrifuged at 2000g for 10 min at 4°C. 3% of the supernatant was set aside as an input. The rest of the supernatant was supplemented with NP-40 to a final concentration of 1% and with DHPC to a final concentration of 30 mM and incubated for 5 min on ice. The samples were centrifuged at 20,000g for 10 min at 4°C, and the supernatant was used for immunoprecipitation with GFP-conjugated beads overnight at 4°C with end-over-end rotation. Subsequently, the beads were washed four times in 0.35 mM KCl TRAP wash buffer (10 mM HEPES-KOH at pH 7.4, 5 mM MgCl2, 350 mM KCl, 1% NP-40, supplemented with 100 µg/mL cycloheximide, 0.5 mM DTT, and 20 U/mL RNasin) and resuspended in 100 µL of RLT buffer from the RNeasy Micro Kit supplemented with 1% β-mercaptoethanol. The resuspended beads were incubated at room temperature for 10 min, placed on a magnet, and the supernatant containing the RNA was collected and purified using the RNeasy Micro Kit, including dsDNAse treatment. RNA integrity was determined using Bioanalyzer RNA 6000 Pico kit (Agilent) prior to RNAseq library preparation. RNA-seq libraries were prepared from 100 ng RNA/sample using the NEBNext Ultra II RNA library preparation kit for Illumina in conjunction with the NEBNext poly(A) mRNA magnetic isolation module and NEBNext multiplex oligos for Illumina. The quality of the sequencing libraries was evaluated using the Agilent 2200 TapeStation with D1000 High-Sensitivity ScreenTape. The samples were sequenced at the Rockefeller University Genomics Resource Center using NovaSeq 6000 to obtain 50-bp single-end reads. Samples with a sufficient number of reads after QC were included in the analysis.