title: GSM6206705: RNA-seq ICM Middle Pregnancy sample4 [M4_ICM]; Homo sapiens; RNA-Seq
library_construction_protocol: Biopsy was performed on the heated stage of an IX-73 microscope (Olympus, Japan), equipped with micromanipulation (Narishige Group, Japan) and an infrared diode laser (Saturn 5TM Laser system; CooperSurgical, Inc., Japan), in dishes prepared with droplets of HEPES-buffered calcium/magnesium-free medium (Sage In-Vitro Fertilization, Inc., USA) and biopsy medium (1062; Origio, Denmark). A diode laser was used to assist the opening of a 10–20 μm hole in the zona pellucida by 2–4 laser shots. ICM biopsy was performed using a glass pipette (PIN20-20FT; PRIME TECH LTD., Japan) either by laser-assisted removal of a small area of the TE layer on the opposite side of the ICM or by directly introducing the biopsy pipette into the blastocoel cavity and aspiration of the ICM cells on the TE layer. TE biopsies were performed by aspirating cells from the TE layer removed during ICM biopsy or newly collected cells from the TE layer after ICM removal using a pipette. In the biopsies used in this study, 10–20 cells were collected for both ICM and TE. Biopsy samples were transferred to a PCR tube (FG-021F; NIPPON Genetics Co., Ltd, Japan) containing 0.5 µL of phosphate-buffered saline (PBS) and frozen. The frozen samples were stored at -80 °C until RNA-seq. RNA-seq libraries of all biopsy samples were generated using a SMART-Seq HT Kit according to the manufacturer's instructions (TAKARA, 634436, Japan). Library construction was performed using a Nextera XT DNA Library Prep Kit (Illumina, FC-131-1024, USA) with 200 pg of cDNA. The libraries were sequenced on a NextSeq 500 with single-end 75-bp reads (Illumina).