GSM7656624: Single oocyte, GV4; Mus musculus; RNA-Seq
To collect the oocytes at fully grown GV stage, around six-weeks-old to eight-weeks-old female mice were injected with 7 IU of pregnant mare serum gonadotropin (PMSG) and humanely sacrificed 44 h later. Then the oocytes at fully grown GV stage were collected in the M2 medium. For collecting the oocytes arrested in second meiotic metaphase (MII stage), approximately 48 h after PMSG (7 IU) injection, 7 IU of human chorionic gonadotropin was injected into female mice. After an additional 20 h, cumulus-oocyte complexes (COCs) were surgically removed from fallopian tubes. Then the oocytes at MII stage were collected in the M2 medium from COCs after digestion of 300 IU/mL hyaluronidase. In the reverse transcription, 2.4 μL of the reverse transcription mix containing 0.8 μL of 5× First Strand Buffer, 0.2 μL of 0.1 M DTT, 0.1 μL of RNaseOUT, 0.15 μL of Superscript III and 1.15 μL of RNase-free water was added into each droplet, and the droplets were treated with ten cycles of annealing (ramping from 8 °C to 50 °C) on the thermocycler. After reverse transcription, the residual primers were digested by T4 polymerase at 37 °C for 40 min and 80 °C for 20 min, and then the RNAs were digested by RNase-H and RNase-If at 37 °C for 15 min and 72 °C for 15 min. Next, dC-tailing and second strand synthesis were performed according to the protocol described in the MATQ-seq technique. The PCR amplification of the droplets were conducted with a 28-cycle PCR program (95 °C for 15 s, 60 °C for 20 s, and 72 °C for 2 min). After PCR amplification, pooled libraries were purified with 1.2X AMPure XP beads into PCR-grade water. The ABclonal Rapid Plus DNA Lib Prep kit of Illumina was used to construct the PCR product library according to the protocol provided with the kit. The cDNA was firstly sheared to 300 bp using a Covaris S220 sonicator. End repair and A-tailed was then performed using a NGS Fast DNA Library Prep kit of Illumina. T4 DNA ligase was used to ligate adaptors to samples. Libraries were then diluted and sequenced on a NovaSeq 6000 machine by Haprolos, using the NovaSeq S4 reagent kit v1.5 (300 cycles).