GSM8586262: TSC_min_DOX_P3_2_input; Homo sapiens; OTHER
For RNA-seq and MeRIP-seq, total RNA was extracted with TRIzol reagent. CUT&Tag was performed according to the manufacturer's protocol (Vazyme, TD904). For ATAC-seq, 50,000 cells were collected and washed twice in pre-cold PBS without DNase. Nuclei were isolated, lysed, and incubated with Tn5 transposase according to the manufacturer's protocol (Vazyme, TD501). Transposed DNA was isolated using a DNA Clean & Concentrator kit (Zymo). For RNA-seq, Next generation sequencing library preparations were constructed according to the manufacturer's protocol (VAHTS mRNA-seq V2 Library Prep Kit for Illumina). The mRNA fragmentation and priming was performed using VAHTSFirst Strand Synthesis Reaction Buffer and VAHTS Random Primers. First strand cDNA was synthesized using ProtoScript II Reverse Transcriptase and the second-strand cDNA was synthesized using Second Strand Synthesis Enzyme Mix. The purified double-stranded cDNA（byAxyPrep Mag PCR Clean-up (Axygen)was then treated with End Prep Enzyme Mix to repair both ends and add a dA-tailing in one reaction, followed by a T-A ligation to add adaptors to both ends. Size selection of Adaptor-ligated DNA was then performed using AxyPrep Mag PCR Clean-up (Axygen), and fragments of ~360 bp (with the approximate insert size of 300 bp) were recovered. Each sample was then amplified by PCR for 11 cycles using P5 and P7 primers, with both primers carrying sequences which can anneal with flow cell to perform bridge PCR and P7 primer carrying a six-base index allowing for multiplexing. The PCR products were cleaned up using AxyPrep Mag PCR Clean-up (Axygen), validated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), and quantified by Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). For MeRIP-seq, The NEBNext® Ultra™ II RNA Library Prep Kit (NEB, E7770) was used to construct libraries for both the input and IP samples. For CUT&Tag, Library preparation and amplification was followed by manufacturer's protocol (Vazyme, TD904). For ATAC-seq, Libraries were prepared by amplifying DNA for 12 cycles using the TruePrep DNA Library Prep Kit (Vazyme). After amplification, libraries were purified using VAHTS DNA Clean Beads (Vazyme).