title: GSM3579343: OSKM-D5-DRIPseq-rep1; Mus musculus; OTHER
library_construction_protocol: For ssDRIP-seq, nucleic were isolated from cells, followed by SDS/proteinase K treatment at 37°C overnight. Genome DNA was extracted by the phenol-chloroform method and precipitated with isopropanol. DNA fragmentation was performed overnight using endonucleases (MboI, MseI, AluI, DdeI), and the negative control was treated with RNase H (New England Biolabs) overnight. Briefly, 3.5 ug of DNA was bound with 10 ug of S9.6 antibody in 1 X binding buffer (10 mM NaPO4 pH 7, 140 mM NaCl, 0.05% Triton X-100) overnight at 4˚C. 50 μl Protein G sepharose beads were added for 4 hours. Bound beads were washed 4 times in binding buffer and elution was performed in elution buffer (50 mM Tris pH 8, 10 mM EDTA, Proteinase K) for 45 min at 55˚C. DNA was purified and sonicated to 150 bp fragments. For RNA-seq, Total RNAs were extracted with TRIzol. For quantitative PCR, cDNAs were synthesized with ReverTra Ace (Toyobo) and oligo dT (Takara), and then analyzed by qPCR with SYBR green mixture (Genstar) and performed on a CFX Real-Time System (Bio-Rad). For ATAC-seq, a total of 50,000 cells were washed once with cold PBS and resuspended in 50 μl lysis buffer. The suspension of nuclei was then centrifuged at 500 g at 4˚C for 10 min, followed by the addition of 50 μl transposition reaction mix (10 μl TD buffer, 5 μl Tn5 transposase and 35 μl nuclease-free H2O). DNA was purified using a MinElute Kit (QIAGEN). For CLIP-seq, mES cells were irradiated at 400 mJ/cm2 with 254 nm UV light. The crosslinked cells were lysed and 15 μg of anti-DDX5 antibody was applied to pull down protein-RNA complexes. After micrococcal nuclease treatment and 3' DNA adaptor ligation, the immunoprecipitated complexes were fractionated on a 4-12% NuPAGE Bis-Tris gel and transferred onto a nitrocellulose membrane. The DDX5-specific smear bands were excised with scalpels and treated with proteinase K (Thermo Fisher Scientific) prior to the extraction of respective RNA by using phenol and chloroform. For ssDRIP-seq, the first adapter was ligated to the 3' end of the ssDNA using Adaptase (Swift Biosciences) via a highly efficient, proprietary reaction that only tails 3' ends of ssDNA and ligates the first truncated adapter to 3' ends simultaneously. This method avoids the bias inherent in random primer-based methods, as it ligates adapters in a sequence-independent manner. The extension step was performed using the primer paired to the first adapter, followed by a ligation reaction to add the second truncated adapter to the 5' ends. An indexing PCR step was performed to add the indexed sequence, and the library was amplified. For RNA-seq, RNA sequencing libraries were constructed using the VAHTS mRNA-seq V3 Library Prep Kit for Illumina (Vazyme). For ATAC-seq, ATAC-seq libraries were first subjected to 5 cycles of pre-amplification. To determine the suitable number of cycles required for the second round of PCR, the library was assessed by quantitative PCR as described, and the library was then PCR amplified for the appropriate number of cycles. Libraries were purified with AMPure XP beads. For CLIP-seq, A 5' RNA linker was then added to the isolated RNA and reverse transcribed by superscript reverse transcriptase III (Life Technologies, 18080-051). The cDNA strands are PCR-amplified to produce libraries for deep sequencing. L3-IR800-biotin DNA adaptor: 5'OH -ATC TCG TAT GCC GTC TTC TGC TTG TAA AAA AAA AAA A/iAzideN/A AAA AAA AAA AA/3Bio/-3', 5' RNA linker: 5'-(OH) rArCrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrU(rN)(rN) (rN)rU(OH) -3'.
attributes.tag: GEO Accession
attributes.value: GSM3579343