GSM5518346: The musk gland -Single-Nucleus RNA-seq; Moschus berezovskii; RNA-Seq
The musk gland sample was quickly put on ice and cut into pieces of about 1-mm3 with scissors. Then it was placed into clean RNase-free Eppendorf tubes, immediately frozen in liquid nitrogen, and stored at -80℃. The frozen tissue was transferred to a 1.5 ml microcentrifuge tube with tweezers, then 500 µl frozen 0.1×lysis buffer was added, and then homogenized 15 times with a particle pestle immediately, and incubated on ice for 5 min. The pipette was mixed with the tip of the large-bore pipette for 10 times and incubated on ice for 10 min. Added 500 µl of freezing wash buffer to the lysed cells. The suspension was passed through a 70 µm filter into a 2 ml test tube, centrifuged at 500 rcf at 4℃ for 5 min, and the supernatant was removed without destroying the nucleus precipitation. Then added 1ml of freezing wash buffer, mixed 5 times with a pipette, centrifuged at 500 rcf at 4℃ for 5 min, and the supernatant was removed without destroying the nucleus precipitation. Finally, the nucleus were resuspended in the cooled diluted nucleus buffer and counted by trypan blue staining. For sNucRNA-seq library, pre-mRNA and mature mRNA were captured using poly (dT) sequence. Subsequently, the GEM structure was broken, all the cDNA were mixed, and PCR amplification was performed to obtain enough cDNA to construct the library. The cDNA were digested into fragments of about 200-300 bp, and then the standard sequencing library was obtained by PCR amplification after conventional next-generation sequencing library construction steps such as end repair, adding A-tailing, sequencing adapters P5, P7 and sample index. High-throughput sequencing was performed on the constructed library using the paired-end sequencing mode of Illumina sequencing platform. In Read 1, a 16 bp Barcode sequence and a 10 bp UMI sequence were included for cell determination and expression quantification; in Read 2, cDNA fragments were included for reference genome alignment to determine the genes corresponding to mRNA.   For sNucATAC-seq library, tagged DNA fragments were captured using spacer sequence. Subsequently, the GEM structure was broken, all the dsDNA were mixed, and PCR amplification was performed to obtain enough DNA to construct the library. Sequencing adapter P7 and sample index were added to DNA fragments by PCR amplification to construct a standard sequencing library. High-throughput sequencing was performed on the constructed library using the paired-end sequencing mode of Illumina sequencing platform. In Read 1, a 16 bp Barcode sequence was included. After excluding the i5 and i7 sample index of 10bp, the remaining sequence information of Read1 and Read2 was used to identify the information of the inserted fragment. sNucRNA-seq and sNucATAC-seq