GSM8849187: Sample LH_2; Ovis aries; RNA-Seq
The single-cell RNA sequencing extraction method is as follows: First, generate GEMs and barcode labeling using the Master Mix, which includes reverse transcriptase reagents, template switch oligos, reducing agent B, and reverse transcriptase enzyme. A total of 33.4 碌L of Master Mix is prepared. Next, load the Master Mix and cell suspension onto the 10x single-cell chip in sequence, distributing them across different wells. Gel Beads and Partitioning Oil are added, and the chip is sealed. Following this, reverse transcription and cDNA amplification are carried out under specific conditions, with the reaction set at 53掳C for 45 minutes. Amplification Reaction Mix is then added for cDNA amplification through PCR, with temperature cycling as follows: 98掳C for 3 minutes, 63掳C for 20 seconds, and 72掳C for 1 minute. Finally, the cDNA is purified and collected to complete the process. These steps ensure efficient single-cell capture and transcriptome analysis. First, DNA fragmentation is performed using appropriate enzymes and buffers to generate small fragments. End repair and A-tailing are then carried out. Next, an adaptor ligation mix is added for adaptor ligation, incorporating sample barcodes. Following this, PCR amplification is performed through the Sample Index PCR step to add sample-specific barcodes. After amplification, the library is purified using SPRIselect Reagents to remove impurities and unligated adaptors. Finally, the library is quality-controlled by measuring concentration with Qubit and performing gel electrophoresis to confirm that the fragment size is within the range of 400-600 bp. The final product is a library ready for high-throughput sequencing.