GSM5460438: 4wk_CD1; Mus musculus; RNA-Seq
Cumulus cells were collected into RLT buffer (1053393, Qiagen, Hilden, Germany) and kept in -80oC until library generation. mRNA was captured using Smart-seq2 oligo-dT pre-annealed to magnetic beads (MyOne C1, Invitrogen, Carlsbad, California, USA). The beads were resuspended in 10 μl of reverse transcriptase mix (100 U, SuperScript II, Invitrogen; 10 U, RNAsin, Promega, Madison, Wisconsin, USA), 1 × Superscript II First-Strand Buffer, 2.5 mM ditiotreitol (DTT, Invitrogen), 1 M betaine (Sigma Aldrich), 9 mM magnesium chloride (MgCl2, Invitrogen), 1 μM Template-Switching Oligo (Exiqon, Vedbaek, Denmark), 1 mM dNTP mix (Roche) and incubated for 60 min at 42 °C followed by 30 min at 50 °C and 10 min at 60 °C (Picelli et al., 2013, 2014). Amplification of the cDNA was then undertaken after adding 11 μl of 2 × KAPA HiFi HotStart ReadyMix and 1 μl of 2 μM ISPCR primer (Picelli et al., 2013, 2014), followed by the cycle: 98 °C for 3 min, then 9 cycles of 98 °C for 15 s, 67 °C for 20 s, 72 °C for 6 min and finally 72 °C for 5 min. Finally, the cDNA was purified using similar volume of AMPure beads (Beckman Coulter, Brea, California, USA) and eluted into 20 μl of water. All libraries were prepared from 100 to 200 pg of cDNA using the Nextera XT Kit (Illumina, San Diego, California, USA), regarding the manufacturer's instructions. The final cDNA library was purified using a 0.7:1 volumetric ratio of AMPure beads before pooling and sequencing.