Antigen

Antigen Class

Unclassified

Antigen

Unclassified

Cell type

Cell type Class

Embryo

Cell type

Morula

MeSH Description

An early embryo that is a compact mass of about 16 BLASTOMERES. It resembles a cluster of mulberries with two types of cells, outer cells and inner cells. Morula is the stage before BLASTULA in non-mammalian animals or a BLASTOCYST in mammals.

Sample

source_name

early embyro

cell type

Morula embyro

strain

BDF1xPWK

treatment

Kdm6bWT injected

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

All animal studies were performed in accordance with guidelines of the Institutional Animal Care and Use Committee at Harvard Medical School. MII-stage oocytes were collected from 8 week-old B6D2F1/J (BDF1) females superovulated by injecting 7.5 I.U. of PMSG (Millipore) and hCG (Millipore). For in vitro fertilization (IVF), MII oocytes were inseminated with activated spermatozoa obtained from the caudal epididymis of adult BDF1 or PWK (Jackson Laboratory, 003715) males in HTF medium supplemented with 10 mg/ml bovine serum albumin (BSA; Sigma-Aldrich). Spermatozoa capacitation was attained by 1 h incubation in the HTF medium. Zygotes were transferred to KSOM and cultured in a humidified atmosphere with 5% CO2/95% air at 37.8°C. RNAseq was performed from whole E3.5 blastocysts (BDF1 X PWK). Each replicate used a pool of 40 embryos. RNA-seq libraries were prepared as previously described (Inoue et al. 2017) with minor modifications. Briefly, reverse transcription and cDNA amplification were performed using whole embryo lysates with SMARTer Ultra Low Input RNA cDNA preparation kit (Clontech, 634890). cDNAs were then fragmented using Tagmentation with Nextera XT DNA library prep kit (Illumina). The fragmented cDNAs were amplified using Nextera PCR master mix according to the manufacturer’s instruction. Single end 100 bp sequencing was performed on a HiSeq2500 sequencer (Illumina). The ULI-NChIP was performed as described previously (Brind’Amour et al. 2015) with the following several modifications. First, we used Beckman SPRIselect beads (Beckman Coulter), instead of Agencourt Ampure XP beads. Second, the sequencing library was prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) according to the manufactures' instruction. Third, PCR amplification was performed using Kapa Hifi hotstart readymix (Kapa Biosystems). Lastly, no size selection step was needed. The library was quantified by Qubit dsDNA HS assay kit (Thermo Fisher Scientific, Q32854) and Agilent high sensitivity assay kit (Agilent Technologies). The libraries were sequenced on a Hiseq2500 with single-end 150 bp reads (Illumina). The H3K27me3 antibody was purchased from Diagenode (C15410069). Before incubation with the antibody, 10% volume of the chromatin lysate was taken for input samples, and the input samples also proceed to the library construction followed by sequencing.

Sequencing Platform

instrument_model

Illumina HiSeq 2500

mm10

Number of total reads

59050589

Reads aligned (%)

77.4

Duplicates removed (%)

87.2

Number of peaks

242 (qval < 1E-05)

mm9

Number of total reads

59050589

Reads aligned (%)

77.3

Duplicates removed (%)

87.3

Number of peaks

202 (qval < 1E-05)