GSM5212238: mESC Wt1 KO, Dox12; Mus musculus; miRNA-Seq
Total RNA was extracted from the cells using the Qiagen miRNeasy column as per the manufacturers protocol Total RNA equivalent to 1µg was ligated with the 3' RNA adapter with the help of the T4 RNA ligase 2 (deletion mutant) followed by ligation of the 5' RNA adapter using T4 RNA ligase. The adapter ligated RNA was then subjected to reverse transcription with Superscript II RT followed by PCR amplification. The amplified cDNA was electrophoresed on a 6% PAGE gel. Mature microRNA, which is approximately 22 bp, gives an adapter ligated fragment of approximately 147 bp. A second band of 157 bp corresponding to piRNAs, other small noncoding RNA arising from 30 bp small RNAs was also excised together to give rise to the pool of small RNA.