Ficoll pm70

Modified M16 medium [10 (link)] was used to culture the embryos. Embryo cryopreservation was performed using 10% (v/v) EG and
10% (v/v) DMSO (Nacalai Tesque, Kyoto, Japan) in FSa solution (EDFS10/10a) (Table 1Table 1.

	EG (% v/v)	DMSO (% v/v)	Sucrose (M)	Ficoll PM-70 (% w/v)	PB1 (% v/v)	Molarity (mol/L)	Molality (moles/kg)
ED5/5	5	5	-	-	90	1.6	2
EDFS10/10a	10	10	0.4	24	56.2	3.2	6.43

EG, ethylene glycol; DMSO, dimethyl sulfoxide.

). The FSa solution was PB1 medium [11 (link)] containing 30% (w/v) Ficoll PM-70 (GE Healthcare, Chicago, USA) and
0.5 M sucrose. The pretreatment solution was PB1 medium containing 5% (v/v) EG and 5% (v/v) DMSO (ED5/5). Permeated cryoprotectants in embryos were removed in
PB1 medium containing 0.5 M sucrose (S-PB1). Unless otherwise noted, chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan).

Cross-linking-free RNA immunoprecipitation was performed as previously detailed (27 (link)), with the addition of a cocktail of molecular crowders [2.5 mg/ml Ficoll PM400, 7.5 mg/ml Ficoll PM70 (both GE Healthcare) and 250 ng/ml Dextran Sulphate 670k (Fluka)] to the immunoprecipitation step. Briefly, 10 μl of antibody [LIN28 (ProteinTech) or U1 (Santa Cruz)] was incubated with BSA-blocked Protein G sepharose beads (Sigma) overnight, washed five times with NT2 buffer, and resuspended in 850 μl of NT2 buffer with molecular buffer cocktail and 200 U of RNase Out and 100 U of SUPERase IN (Life Technologies). Human ESC cultures that were 50–70% confluent (1–5 × 10⁶ cells) were then washed with phosphate buffered saline (Invitrogen) and lysed with 500 μl of polysome lysis buffer. Nuclei were removed by centrifugation at 5000 × g for 5 min. 100 μl of the cytoplasmic lysate was then added to 900 μl of antibody-coated bead slurry from above for each sample, and rotated at 4°C overnight. The next day, beads were washed five times with NT2, and the RNA eluted with Trizol reagent (Invitrogen), followed by standard RNA precipitation protocols. The RNA was resuspended in 50 μl of RNase-free water and ready for either real-time PCR or microarray analysis, where volumes were kept constant across all samples. 10 μl of RNA was used for each sample for preparation of cRNA for microarray analysis.