title: GSM8520915: NSC-Uli-epic-BIDSeq-input-rep1; Mus musculus; OTHER
library_construction_protocol: HEK293T total RNA was extracted by Trizol in accordance with the manufacturer's protocol. mRNA were purified by VAHTS mRNA capture beads (Vazyme).    Sperm were collected from the cauda epididymis of 10-12 week old mice and released into 2 mL of phosphate-buffered saline (PBS) maintained at 37°C for a 20-minute incubation. Following incubation, the suspension was filtered through a 40 µm cell strainer to remove tissue debris. Subsequently, the sperm were treated with 10 mL somatic cell lysis buffer (0.1% SDS, 0.5% Triton X-100) on ice for 40 minutes to eliminate somatic cell contamination. The purity of the sperm sample was confirmed via microscopic examination. The sperm pellet was washed twice in 5 mL of PBS, followed by centrifugation at 1000g for 5 minutes. The sperm pellet was resuspended in TRIzol reagent (Invitrogen), homogenized, and subjected to RNA extraction. The hippocampi were isolated from embryonic 17.5 Nestin-GFP mice and dissociated using the Neural Tissue Dissociation Kit P (Miltenyi Biotec, 130-092-628). Following enzymatic digestion, the solution was diluted by adding 9 mL of Hibernate™-A medium (Invitrogen, A1247501) and centrifuged at 250 × g for 8 minutes. The cell pellets were then resuspended in 0.5 mL of 1× red blood cell lysis buffer (Biogems, 64010-00-100) and incubated for 1 minute. Subsequently, 5 mL of Hibernate™-A medium was added, and the mixture was centrifuged again at 250 × g for 6 minutes. The cells were resuspended in FACS sorting medium (0.5% BSA in Hibernate™-A), incubated with DAPI (Solarbio, C0060) at a 1:1,000 dilution for 5 minutes to label dead cells, and passed through a cell strainer. Finally, the cells were sorted using a BD FACS Aria II cell sorter. Total RNAs were isolated from sorting cells by using TRIzol reagent (Invitrogen) and MaXtractTM High Density (Qiagen). Uli-Epic BID-seq steps are combined steps of our uli-epic and reported bulk BID-seq. 1 ng or 100 pg 293T mRNA and were fragmented using 0.1M NaHCO3 pH 9.2 and incubate at 95 °C for 5 min, then add 3M NaOAc pH 5.2 to neutralize pH. RNA was 3' end-repaired by T4 polynucleotide kinase (PNK) (catalog no. M0201, NEB). Then add poly (A) tailing to the 3' end of RNA by E. coli Poly(A) Polymerase (NEB, M0276L). Carrier DNA was added to poly (A) RNA and was purified by Oligo Clean & Concentrator（Zymo Research，D4061), eluting with 7 µl RNase-free water. A 1.5 µl aliquot of the purified RNA was saved for 'Input' library construction, 5 µl was subjected to BID-seq optimized bisulfite treatment, as the 'Treated' sample. The 5 µl RNA was mixed with 45 µl freshly prepared BID-seq BS reagent (2.4 M Na2SO3 and 0.36 M NaHSO3, prepared by dissolving 270 mg Na2SO3 and 34 mg NaHSO3 in 850 µl RNase-free water) and incubated at 70 °C for 3 h. Then, 100 µl RNase-free H2O, 250 µl RNA binding buffer (RNA Clean and Concentrator), and 400 µl ethanol were added to the reaction mixture, which was mixed well and loaded on a RNA Clean and Concentrator-5 column. After spinning and washing once with 200 µl RNA wash buffer (RNA Clean and Concentrator), 200 µl RNA Desulphonation Buffer (catalog no. R5001-3-40, Zymo Research) was added to the column and incubated at room temperature for 1 h. This was followed by spinning and washing twice with 700 µl RNA wash buffer (RNA Clean and Concentrator), followed by eluting RNA with 10.5 µl RNase-free water. Reverse transcription and template switching were performed with template switching RT enzyme mix (NEB, M0466L)、 T7 primer (sequence: 5'-GCCGCGAAATTAATACGACTCACTATAGGGATAATGAGCAGACGTGTGCTCTTCCGATCTTTTTTTTTTTTTTTTTTTTTVN-3') and Template switch oligo (TSO: 5'Biotin-GCTAATCATTGCACACGACGCTCTTCCGATCTrGrGrG-3') at 42 °C for 90 min. Then RNA template was degradation by E. coli RNase H (NEB, M0297L), followed by second strand cDNA were synthesized by Q5 Hot Start High Fidelity Master Mix (NEB #M0494). The excess T7 primer was digested by exonuclease I (NEB, M0293S). The double strand cDNA was purified by Oligo Clean & Concentrator (Zymo Research，D4061). In vitro transcription was carried out with double strand cDNA by T7 High Yield RNA Transcription Kit, vazyme. At the end of in vitro transcription, DNase I was added to digest double strand cDNA. RNA was purified by 1.8 × SPRIselect beads (Beckman, B23318). Then the RNA was subjected to reverse transcription with RT primer (sequence: 5'- ACACGACGCTCTTCCGATCT -3') by SuperScript™ III (Thermo Fisher Scientific, cat. no.18080051). cDNA was purified with 1.8 × SPRIselect beads (Beckman, B23318). Library were constructed with single index primers (NEB, E7335S).