GSM9128243: CCDC134, Input, rep2; Homo sapiens; OTHER
All buffers were supplemented with 100 ng/mL cycloheximide. Microsomes were isolated from nine 15-cm dishes. After treatment with 10,000 U of micrococcal nuclease for 30 min at 37 °C, microsomes were pelleted and solubilized with digitonin. Ribosomal complexes in the soluble microsome fraction were isolated by ultracentrifugation through a sucrose cushion. The ribosomal pellet was resuspended and used as input. The elution following immunoprecipitation from M2 Flag resin was used as the IP fraction. To prepare RNase footprints, input and IP samples were digested with RNase 1 at 0.5 - 1 U/µL within a 90 µL reaction at room temperature for 1.5 h. RNase footprints were extracted by adding 400 µl of TRIzol, followed by 100 µl of chloroform. RNA in the aqueous layer was separated by centrifugation at 12000 g for 15 min at 4 ℃ and then precipitated overnight by adding 0.1 volume of 3 M sodium acetate, 10 mg of glycoblue, and 1.2 volumes of isopropanol. Purified RNase footprints were used for library preparation. RNase footprints were resuspended in 5 µl PNK reaction mixture (1x PNK buffer, 10 U PNK (NEB), and 10 U SUPERase·In) and end-repaired at 37 ℃ for 1 h. The reaction was added with 5 µl of polyadenylation mix containing 10 U E. coli poly (A) polymerase (NEB), 2 µl of 5x first-strand buffer (250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2), 0.25 mM ATP, and 10 U SUPERase·In and incubated at 37 ℃ for 2 h. The resulting polyadenylated RNA was then reverse transcribed by a SMART-RT reaction. The reaction mixture was first added with 2.5 µl of RT primer (10 µM, 5'-ACGTGTGCTCTTCCGATCTNNNNNNNNNNNNNNNTTTTTTTTTTTTTTTVN-3', where “N” is any base and “V” is either “A”, “C” or “G”) and annealed by heating to 72 °C for 5 min and cooled on ice, and then supplemented with SMAT-RT mixture (2.5 µl dNTP (10 mM), 400 U Superscript II reverse transcriptase (Invitrogen), 10 U SUPERase·In, 4 µl Superscript II first-strand buffer (5x), 1.5 µl DTT (100 mM), 6 µl Betaine (5 mM), 0.12 µl MgCl2 (1 M), 0.5 µl template-switching oligos (100 µM 5'-CTCTTTCCCTACACGACGCTCTTCCGATCTNNNNrGrG+G-3', where “N” is any base and “+G'' is a locked nucleic acid guanylate), and 0.5 µl nuclease-free water. Reverse transcription was carried out with incubation at 42 ℃ for 1 h, followed by 10 cycles of heating at 50 °C for 2 min and then incubating at 42 ℃ for 2 min. The reverse transcriptase was inactivated by heating at 70 ℃ for 10 min. RNA footprints hybridized to DNA were removed by incubating at 37 ℃ for 15 min with 5 U of RNase H. The resultant cDNA was amplified by a first round of PCR with 7 cycles, purified, and then amplified by a second round of PCR with 4 cycles. The DNA library was visualized and separated in a 4% agarose gel. DNA with a 15-35 bp insert was excised and recovered using a DNA gel recovery kit (Zymo). Equal amounts of barcoded libraries were pooled and sequenced with Illumina NovoSeq.