title: GSM4817813: E4_5_MUT.MUT_19; Mus musculus; RNA-Seq
library_construction_protocol: For E3.5 blastocysts, zona pellucidae were removed using acid tyrode's solution (Sigma) and embryos subjected to immunosurgery using 20% anti-mouse whole antiserum (Sigma) in N2B27 in at 37oC, 7% CO2 for 30 minutes, followed by 3 rinses in M2, then 15 minutes in 20% non-heat inactivated rat serum (made in house) in N2B27 in at 37oC, 7% CO2. After 30 minutes in fresh N2B27 lysed trophectoderm was removed and placed in lysis buffer for genotyping. ICMs were incubated in 0.025% trypsin (Invitrogen) plus 1% chick serum (Sigma) for 5-10 minutes in small drops and dissociated by repetitive pipetting using a small diameter mouth-controlled flame-pulled Pasteur pipette. Individual ICM cells were transferred into single cell lysis buffer and snap frozen on dry ice. Smart-seq2 libraries were prepared as described previously and sequenced on the Illumina platform in a 125 bp paired-end format.