Human embryos were obtained from two cohorts at the Huddinge Karolinska Hospital and Carl von Linné Clinic with ethical approval from regional ethics board (2012/1765-31/1). The first cohort was from preimplantation genetic diagnosis (PGD) testing on embryonic day (E) 4 and cultured until E7 (expanded blastocyst, just prior to implantation) under standard conditions as performed in the IVF Clinic (5% CO2/5% O2 in CCM media (Vitrolife) covered with Ovoil (Vitrolife). The second cohort was from frozen E2 embryos thawed (ThawKit Cleave, VitroLife) and cultured in G-1 Plus media (VitroLife) and from E3 in CCM media. As we are restricted to embryos cultured in vitro, we cannot exclude potential differences with their in vivo counterparts. However, we anticipate these differences to be relatively subtle as in vitro cultured embryos used in infertility treatment progress and give rise to viable offspring.
Embryos were dissociated through trituration in TrypLE, (Life Technologies) and picked with fine glass capillaries. For a subset of E5–E7 embryos, ICM cells were enriched using immunosurgery (15 embryos). Cells were dispensed in lysis buffer, and cDNA libraries were generated using Smart-seq2 (Picelli et al., 2014 (link)). Briefly, following cell lysis, PolyA(+) RNA was reverse transcribed using SuperScript II reverse transcriptase (Invitrogen) and nested primers, utilizing a strand-switch reaction to add a reverse primer for the second-strand synthesis. The cDNA was amplified by PCR (18 cycles) using KAPA HiFi HotStart ReadyMix (KAPA Biosystems) and purified using magnetic beads. The quantity and quality of the cDNA libraries were assessed using an Agilent 2100 BioAnalyzer (Agilent Technologies). cDNA (∼1 ng) was tagmented using transposase Tn5 and amplified with a dual-index (i7 and i5; Illumina; 10 cycles) and individual Nextera XT libraries were purified with magnetic beads. Indexed sequence libraries were pooled for multiplexing (∼40 samples per lane), and single-end sequencing was performed on HiSeq 2000 using TrueSeq dual-index sequencing primers (Illumina). For further details and data analysis see theSupplemental Experimental Procedures .
Embryos were dissociated through trituration in TrypLE, (Life Technologies) and picked with fine glass capillaries. For a subset of E5–E7 embryos, ICM cells were enriched using immunosurgery (15 embryos). Cells were dispensed in lysis buffer, and cDNA libraries were generated using Smart-seq2 (Picelli et al., 2014 (link)). Briefly, following cell lysis, PolyA(+) RNA was reverse transcribed using SuperScript II reverse transcriptase (Invitrogen) and nested primers, utilizing a strand-switch reaction to add a reverse primer for the second-strand synthesis. The cDNA was amplified by PCR (18 cycles) using KAPA HiFi HotStart ReadyMix (KAPA Biosystems) and purified using magnetic beads. The quantity and quality of the cDNA libraries were assessed using an Agilent 2100 BioAnalyzer (Agilent Technologies). cDNA (∼1 ng) was tagmented using transposase Tn5 and amplified with a dual-index (i7 and i5; Illumina; 10 cycles) and individual Nextera XT libraries were purified with magnetic beads. Indexed sequence libraries were pooled for multiplexing (∼40 samples per lane), and single-end sequencing was performed on HiSeq 2000 using TrueSeq dual-index sequencing primers (Illumina). For further details and data analysis see the
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