title: GSM8993191: TMCO1, Input, rep2; Homo sapiens; OTHER
library_construction_protocol: Cells were lysed, and microsomes were isolated. After treatmnet with 10,000 U micrococcal nuclease for 30 min at 37 °C, microsomes were pelleted and solubilized. The cleared supernatant was used as input, after M2 Flag affinity beads binding, immunoprecipitated material was used as IP. 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 RT for 1.5 h. RNase footprints were extracted by adding 400 µl 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 TNK 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 SMAT-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 then amplified by a first round of PCR with 7 cycles. Purified PCR1 products were amplified by a second round of PCR with 4 cycles. The DNA library was visualized and separated in a 4% agarose gel. DNA with 15-35 bp-insert was excised and recovered using a DNA gel recovery kit (Zymo). Equal amounts of barcoded libraries were pooled and then sequenced with Illumina NovoSeq.