GSM9598940: intTex_RNA_KO_3; Mus musculus; RNA-Seq
For ATAC-seq, the extracted nuclei were re-suspended fragmented buffer including 1.5 μL transposome (novoprotein N248), and incubated at 37 oC for 30 min. For CUT&Tag, cells were sorted enriched by ConA-magnetic beads, and then incubated overnight with antibody. The next day, beads were washed and incubated with anti-rabbit secondary antibody. Then the beads were washed and incubated with proteinA/G-Tnp transposome in Chitag. Next, cells were resuspended in 100 μL Tagmentation buffer (10 mM MgCl in Chitag Buffer) and incubated at 37 °C for 1 hr. The tagmentation was terminated by adding 1 μL of 10% SDS at 55 °C for 10 min. For HiC, the sorted cells were crosslinked with 1% formaldehyde and lysed in 1 ml lysis buffer. The resulting chromatin was then digested with NUB I (NEB) restriction enzymes at at 37 °C overnight. The DNA ends were blunted and labelled with biotin by Klenow DNA polymerase, followed by ligation using T4 DNA ligase (NEB). After reverse crosslinking, DNA was fragmented by sonication. For RNA-seq, RNA was extracted using the Direct-zol RNA MiniPrep kit (Zymo Research). A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations.For m3C-seq, RNA samples were subjected to alkaline hydrolysis for fragmentation. RNA fragments were dephosphorylated with antarctic phosphatase (NEB, USA) and then incubated in 1M aniline for cleavage. For RIP-seq, the RNA immunoprecipitation assay was carried out with GenSeq RIP kit (GenSeq Inc.) according to the manufacturer's instructions. rRNAs were removed by using GenSeq rRNA Removal Kit (GenSeq, Inc.). For ATAC-seq, the products were purified with tagment DNA extract beads (novoprotein N245), and then amplified by PCR for 14 cycles with NovoNGS index kit for illumine (N239). For CUT&Tag, the DNA fragments added 1 μL spike in (Vazyme) and were extracted by DNA extract beads, then amplified by PCR for 12 cycles with TruePrep index kit for illumine. For HiC, the DNA fragments were then end-repaired and the biotinylated DNA fragments were captured using Dynabeads MyOne Streptavidin C1 beads (Invitrogen, Termo Fisher Scientifc). The DNA on beads was ligated to the Truseq adaptors and amplifed with PCR for library construction. For RNA-seq, Sequencing libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB) following manufacturer's recommendations.For m3C-seq, RNA libraries were constructed with GenSeq® Small RNA Library Prep Kit (GenSeq Inc.) by following the manufacturer's instructions.For,RIP-seq, RNA libraries were constructed by using rRNA-depleted RNAs with GenSeq Low Input RNA Library Prep Kit (GenSeq, Inc.) according to the manufacturer's instructions.