title: GSM7791821: Sample4_Treg spinal cord 1; Mus musculus; RNA-Seq
library_construction_protocol: 2.1.1 Transfer appropriate tissue sample into corresponding tissue grinding tube (tissue≤30 mg, cell≤1×107). 2.1.2 Add 600 μL Buffer RLT Plus. Homogenize the lysate for using a rotor–stator homogenizer, stay at room temperature for 5 min. 2.1.3 Centrifuge at 12,000×g for 3 min in room temperature, transfer the supernatant to AllPrep DNA spin column. 2.1.4 Centrifuge 12,000×g for 30 s in room temperature, transfer the solution from the collection tube for the RNA purification. 2.1.5 Transfer the DNA spin column to the new collection tube for DNA purification.                                                                                                             2.3.1 Add the equal volume of 70% ethanol to the collection tube, mix by pipetting. 2.3.2 Transfer 700 μL mixed solution, to a RNeasy spin column placed in a 2 mL collection tube. 2.3.3 Centrifuge at 10,000×g for 30 s and allow it to empty by gravity flow. 2.3.4 Add 700 μL Buffer RW1 to RNeasy spin column. Centrifuge at 10,000×g for 30 s and discard the filtrate. 2.3.5 Add 500 μL Buffer RPE to RNeasy spin column. Centrifuge at 10,000×g for 30 s and discard the filtrate. 2.3.6 Add 500 μL Buffer RPE to RNeasy spin column. Centrifuge at 10,000×g for 30 s and discard the filtrate. 2.3.7 Centrifuge at 16,000×g for 2 min and transfer the RNeasy spin column to the 1.5 mL centrifuge tube. 2.3.8 Add 35 μL RNase-free water to RNeasy spin column. Stay at room temperature for 1min. 2.3.9 Centrifuge at 16,000×g for 2 min at room temperature to collect RNA. 2.Experimental Procedure 2.1 Sample QC Select the corresponding testing methods for quality inspection according to the requirements of samples and products. 2.2 mRNA Isolation A certain amount of RNA samples are denatured at suitable temperature to open their secondary structure, and mRNA is enriched by oligo (dT) -attached magnetic beads. 2.3 mRNA Fragmentation The reaction system is configured. After reacting at the suitable temperature for a fixed period of time, RNAs are fragmented. 2.4 cDNA Synthesis Prepare the first-strand synthesis reaction system, and set up the reaction program, synthesize the first- strand cDNA, prepare the second-strand synthesis reaction system, and set up the reaction program to synthesize the second-strand cDNA. 2.5 End Repair, Add A and Adaptor Ligation After the reaction system and program are configured and set up, double-stranded cDNA fragments are subjected to end-repair, and then a single 'A' nucleotide is added to the 3' ends of the blunt fragments. The reaction system and program for adaptor ligation are subsequently configured and set up to ligate adaptors with the cDNAs. 2.6 PCR The PCR reaction system and program are configured and set up to amplify the product. 2.7 Library QC The corresponding library quality control protocol will be selected depending upon product requirements. 2.8 Circularization Single-stranded PCR products are produced via denaturation. The reaction system and program for circularization are subsequently configured and set up. Single-stranded cyclized products are produced, while uncyclized linear DNA molecules are digested. 2.9 Sequencing Single-stranded circle DNA molecules are replicated via rolling cycle amplification, and a DNA nanoball (DNB) which contain multiple copies of DNA is generated. Sufficient quality DNBs are then loaded into patterned nanoarrays using high-intensity DNA nanochip technique and sequenced through combinatorial Probe-Anchor Synthesis (cPAS).