GSM6415638: SCI2: spinal cord, injuried, 3 days; Mus musculus; RNA-Seq
A. Take an appropriate amount of tissue/cell sample, add 600 μL Lysis/Binding Buffer, and homogenize. Add 30 μL miRNA  Homogenate Additive, mix well. Ice bath for 10 min; B. Add an equal volume of phenol (acid phenol) chloroform - self-preparation. Centrifuge at maximum speed for 5 min at room temperature. Take the supernatant; C. Add 1.25 times the volume of 100% ethanol; D. Add the mixture to the column, (note: add up to 700 μL) at room temperature, 13000 rpm, 30 s, discard the supernatant; E. Add 350 μL of miRNA Wash Solution 1 to the spin column, 13000 rpm for 30 s. Discard the supernatant and leave  The stem is repositioned into the collection tube; F. Mix 10 μL DNase I and 70 μL Buffer RDD QIAGEN (#79254) Total volume: 80 μL, add  Put it on the membrane in the spin column and leave it at room temperature for 15 minutes; G. Add 350 μL of miRNA Wash Solution 1 to the spin column, 13000 rpm for 30 s. Discard the supernatant and leave  The stem is repositioned into the collection tube; H. 500 μL Wash Solution 2/3 was passed through the column twice, 13000 rpm, 30 s. Discard the supernatant and replace the spin column  Placed in the collection tube; I. Centrifuge the empty column for 1 minute. Place the spin column in a new collection tube and add 100 μL of pre-warmed 95°C to the center of the column.  Elution Solution for 2 min. Centrifuge at the highest speed for 20-30 seconds at room temperature, the liquid in the collection tube is the extracted liquid  Total RNA can be stored at -70°C. 1. Purification and fragmentation of mRNA 2.Synthesis of cDNA 1 strand 3.Synthesis of cDNA 2 strands 4.Plus A at the 3 prime end 5.The connection of the joint sequence 6.Enrichment of DNA fragments 7. Library quality inspection