GSM8969139: MII_longRNA-seq_4; Homo sapiens; RNA-Seq
Total RNAs were extracted from mouse oocytes and cultured cells using TRIzol reagent (Ambion), respectively. The quantity of isolated RNA was measured with Qubit 2.0 Fluorometer (Invitrogen). Lysates from the single oocyte were incubated at 72 °C for 3 minutes to release and unfold the small RNAs. After 3′ adapter ligation, 5 U of lambda exonuclease and 25 U of 5′ deadenylates were used to remove the excess 3′ adapter. Subsequently, 5′ adapter was ligated, followed by reverse transcription and pre-amplification. For final amplification, 1 μL of the pre-amplified product was used as a template. Libraries were size-selected by electrophoresis on a 6% polyacrylamide gel, and 130–160 bp DNA fragments were extracted to recover small RNA libraries. Single-oocyte mRNA was captured and amplified using Smart-seq2, as previously described, 69. 2 ng of purified cDNA was subjected to a tagmentation reaction with Tn5 transposase70.