title: GSM8520924: FGR-NSC-Uli-epic-GLORI-rep2; 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 GLORI steps are combined steps of our uli-epic and reported bulk GLORI. In brief, 10ng 293T mRNA or 10ng mouse sperm ribo-depletion RNA or 10ng mouse NSC ribo-depletion RNA was added into 40 μL of reaction buffer containing 6 μL of glyoxal solution (Sigma-Aldrich, 50649), 20 μL of DMSO and nuclease-free water. The mixture was incubated at 50 °C for 30 min. Then 10 μL of saturated H3BO3 (Sigma-Aldrich, B0394) solution was added and the incubation continued at 50 °C for 30 min followed by cooling on ice for further use. The 50 μL of reaction mixture was added to premixed deamination buffer and gently mixed. The tubes were incubated for 8 h at 16 °C. RNA was purified by Oligo Clean & Concentrator (Zymo Research，D4061). Then RNA were dissolved by 50 μL deprotection buffer(文献). The mixture was incubated at 95 °C for 10 min. RNA was purified with Oligo Clean & Concentrator（Zymo Research，D4061）, and then end-repaired by PNK (NEB, M0201S) to free the 3' OH. Then add poly (A) tailing to the 3' end of RNA by E. coli Poly(A) Polymerase (NEB, M0276L). RNA was purified by Oligo Clean & Concentrator（Zymo Research，D4061). 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-GCTAATCATTGCACACGACGCTCT TCCGATCTrGrGrG-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).