GSM8759165: RNA-seq_rep_sgRunx3_P14_Cl13_D5.5_1; Mus musculus; RNA-Seq
A total of 1 × 10⁵ donor-derived P14 cells were sorted and lysed in lysis buffer (100 mM Tris-HCl, pH 7.5; 500 mM LiCl; 10 mM EDTA, pH 8; 1.5% SDS) containing oligo-dT magnetic beads conjugated with Oligo-dT25. The mixture was incubated for five minutes to facilitate mRNA binding. Subsequently, the sample was placed on a magnetic rack to separate the beads, and the supernatant was discarded. The beads were washed twice with Wash Buffer A (10 mM Tris-HCl, pH 7.5; 0.15 M LiCl; 1 mM EDTA, pH 8; 0.1% SDS) and once with Wash Buffer B (10 mM Tris-HCl, pH 7.5; 0.15 M LiCl; 1 mM EDTA, pH 8) to remove non-specific contaminants. The supernatant was carefully removed after each wash. Elution of mRNA was achieved by adding 5 μL of Elution Buffer (10 mM Tris-HCl) and incubating the beads at 80°C for two minutes. The mixture was then subjected to magnetic separation, and the supernatant containing the extracted mRNA was collected. The isolated RNA was reverse transcribed into cDNA using a reaction mixture containing dNTPs (Transgen, AD101), oligodT mix, Maxima H Minus Reverse Transcriptase (Thermo Fisher, EP0753), and RNase inhibitor (Thermo Fisher, EO0382). A template switch primer was added for synthesizing the second strand of cDNA, followed by 11 cycles of amplification. The DNA fragments were then fragmented, and the library was constructed according to the manufacturer's procedure using the TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme). The library was purified and subjected to paired-end sequencing (2 × 150 bp) on an Illumina NovaSeq platform.