title: GSM8630740: mmem_ctl_sl050; Mus musculus; RNA-Seq
library_construction_protocol: To prepare cell lysates, we first create a 1% solution of 2-mercaptoethanol in TCL buffer. We distribute 5 μl of this solution into each well of a skirted-side 96-well PCR plate, placing a single embryo in each well. The plate is then centrifuged for 1 minute at 800 × g at room temperature. Next, we add 10 μl of RNase-free H₂O to each lysate, followed by 2.2 volumes of RNA-SPRI beads. The lysates are incubated for 10 minutes on the bench to allow for RNA binding, and then the plate is placed on a 96-well plate magnet and incubated for 5 minutes to separate the beads. After carefully removing the supernatant, the beads are washed by adding 75 μl of 80% ethanol to each well. The plate is left to air dry at room temperature for 10 minutes. Finally, the RNA is eluted by adding 4 μl of RNase-free H₂O to each well. To reverse transcribe mRNA, prepare the workspace, then elute RNA with a primer and dNTP mix. After initial incubation and centrifugation, add a mastermix with SuperScriptII buffer, DTT, betaine, MgCl₂, a template-switching oligo, RNase inhibitor, and reverse transcriptase. Reverse transcribe with a thermal cycler protocol (42°C for 90 mins, cycles at 50°C/42°C, then 70°C). For amplification, add KAPA HiFi mix and PCR primer, then amplify with 20 PCR cycles and store overnight at 4°C. Clean up with DNA SPRI beads, washing with ethanol, then elute in TE buffer. Measure size and concentration, then proceed with Nextera XT library prep: perform tagmentation, add NT Buffer, indexed primers, and amplify. Pool samples, perform SPRI bead cleanup, and measure for sequencing readiness. single embryo mRNA-seq