title: Illumina HiSeq 2000 sequencing; RNA targets of FASTKD2 in Human HEK293 cells
design: RNA targets of FASTKD2 in Human HEK293 cells
library_construction_protocol: Cells were cultured at 37C, 95 % humidity and 5 % CO2 in 1 ?? high glucose DMEM supplemented with 10 % fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin and 100 ??g/mL streptomycin sulfate. Induced HEK293 Flp-In TRex cell lines expressing FASTKD2-GFP (10 ng/mL tetracyclin, 16 hrs) were washed twice with PBS and irradiated with UV light at 254 nm (150 mJ/cm2) on ice. Immediately after irradiation cells were lysed in three cell pellet volumes of lysis buffer (100 mM KCI, 5 mM MgCl2, 10 mM Tris-HCI pH 7.5, 0.5 % NP-40, 4 mM DTT, 100 U/mL RNasin [Promega], 200 ??M ribonucleoside vanadyl complex [NEB], EDTA-free protease inhibitor cocktail [Roche]). Lysates were cleared by centrifugation at 16 000 x g for 20 min at 4C. Cleared lysates (typically 2 mL) were then mixed 1:4 with 5 ?? High Salt Buffer (1.25 M NaCl, 100 mM Tris-HCl pH 7.5, 0.5 % sodium dodecyl sulphate). The lysates were pre-cleared by incubation with 60 ??L of equilibrated blocked magnetic particle suspension (Chromotek) per mL of lysate (1 h, 4C, gentle rotation). GFP fusion proteins were then captured from pre-cleared lysates by incubation with 60 ??L of GFP-Trap magnetic particle suspension (GFP-Trap_M, Chromotek) per mL of lysate (2 h, 4C, gentle rotation). Beads were collected using a magnet and washed twice with high salt buffer. The beads were then washed twice with medium salt buffer (250 mM NaCl, 20 mM Tris-HCl pH 7.5, 1 mM MgCl2, 0.05 % NP-40, 100 U/mL RNasin, 200 ??M ribonucleoside vanadyl complex, EDTA-free protease inhibitor cocktail) and once with low salt buffer (150 mM NaCl, 20 mM Tris-HCl pH 7.5, 1 mM MgCl2, 0.01 % NP-40, 50 U/mL RNasin, EDTA-free protease inhibitor cocktail). Beads were resuspended in 1 mL low salt buffer without RNasin and incubated (3 min, 37C, 1 100 rpm) with 4 U of RNAse I (Life Technologies) and 4 U of Turbo DNase (Life Technologies). Beads were washed once with high salt buffer, twice with low salt buffer, re-suspended in 20 ??L of PNK mix (70 mM Tris-HCl pH 6.5, 10 mM MgCl2, 5 mM DTT, 250 U/mL T4 polynucleotide kinase [NEB], 500 U/mL RNasin) and incubated with agitation (1 100 rpm) for 20 min at 37C. Beads were then washed twice with high salt buffer, twice with low salt buffer and incubated for 16 h (1 100 rpm, 16C) in 20 ??L ligation mix (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 10 mM DTT, 500 U/mL T4 RNA ligase 1, 500 U/mL RNasin, 1.5 ??M pre-adenylated linker L3 [5???-rApp-AGA TCG GAA GAG CGG TTC AG-ddC-3???], 20 % PEG-400 [Sigma]). The beads were washed three times with high salt buffer, twice with low salt buffer, proteins digested with proteinase K and samples processed for all subsequent steps as described previously (Nat Struct Mol Biol 17:909-915, 2010.). cDNA libraries obtained after PCR amplification with universal Solexa primers (25 cycles) were multiplexed and sequenced using an Illumina HiSeq2000 platform.
attributes.tag: Experimental Factor: FlpInTREx_MLS-EGFP
attributes.value: Genotype