GSM9455737: m4 Padi6 mutant blastocyst; Mus musculus; RNA-Seq
DNA and RNA were separated using the G&T protocol [59]. Magnetic beads (MyOne C1, Life Technologies) were washed and annealed to oligo dTs and then used to capture polyadenylated mRNA from individual cell lysates. The supernatant containing the DNA was transferred to a new tube, and the beads washed three times in 1× FSS buffer (SuperScript II, Invitrogen), 10 mM DTT, 0.005% Tween-20 (Sigma), and 0.4 U/μl RNasin (Promega) to remove all DNA residues. Each washing solution was added to the DNA tube to maximize recovery. The mRNA bound to the beads was processed further for cDNA conversion. This involved the resuspension of the beads in 10 μl of reverse transcriptase master mix (100 U of SuperScript II [Invitrogen], 10 U of RNasin [Promega], 1× Superscript II first-strand buffer, 5 mM DTT [Invitrogen], 1 M betaine [Sigma], 9 mM MgCl2 [Invitrogen], 1 μM template-switching oligo [TSO; Eurogentec], 1 mM dNTP mix [Roche]). The mRNA mixture was reverse-transcribed by incubation for 60 min at 42°C followed by 30 min at 50°C and 10 min at 60°C. The cDNA obtained was subjected to PCR amplification by adding 11 μl of 2× KAPA HiFi HotStart ReadyMix and 1 μl of 2 μM ISPCR primer. The amplification was performed in a thermocycler for 3 min at 98°C, followed by 17 cycles of 15 sec at 98°C, 20 sec at 67°C, and 6 min at 72°C, and a final extension step of 5 min at 72°C. The amplified product was purified using Ampure XP beads with a 1:1 ratio and eluted into 20 μl of water. Libraries were prepared from 100–400 pg of cDNA using the Nextera XT kit (Illumina) following the manufacturer's instructions but with one-fifth volumes. All 20 single-blastocyst RNA-seq libraries were pooled together and sequenced on an Illumina NovaSeq X platform to an average depth of 4.2 million reads using paired-end 75-bp read length settings.