GSM9157755: PBS; Mus musculus; RNA-Seq
Nuclei were isolated from injured mouse spinal cords at day 7 post-injury for snRNA-seq. Briefly, mice were euthanized and spinal cord was rapidly dissected and frozen in liquid nitrogen. The nucleus was isolated using IGEPAL® CA-630 (Sigma I8896) as instructions. The samples were cut and transferred to a 5 mL tube containing lysate, mixed and lysed for 2 min on ice, then filtered through a 40 μm filter. Nucleus count and viability was estimated using SeekMate Tinitan Fluorescence Cell Counter (SeekGene M002C) with AO/PI reagent. Subsequent experiments were performed if the nuclear envelope was intact and there were fewer impurities. libraries were prepared using SeekOne® DD Single Cell 3' library preparation kit (SeekGene). Briefly, appropriate number of cell nucleus were mixed with reverse transcription reagent and then added to the sample well in SeekOne® DD Chip S3. Subsequently Barcoded Hydrogel Beads (BHBs) and partitioning oil were dispensed into corresponding wells separately in chip S3. After emulsion droplet generation reverse transcription were performed at 42 ℃ for 90 minutes and inactivated at 85 ℃ for 5 minutes. Next, cDNA was purified from broken droplet and amplified in PCR reaction. The amplified cDNA product was then cleaned, fragmented, end repaired, A-tailed and ligated to sequencing adaptor. Finally, the indexed PCR were performed to amplified the DNA representing 3' polyA part of expressing genes which also contained Cell Barcode and Unique Molecular Index. The indexed sequencing libraries were cleanup with VAHTS DNA Clean Beads (Vazyme, N411-01), analyzed by Qubit (Thermo Fisher Scientific, Q33226) and Bio-Fragment Analyzer (Bioptic, Qsep400). The libraries were then sequenced on illumina NovaSeq X Plus with PE150 read length.