Antigen

Antigen Class

Input control

Antigen

Input control

Cell type

Cell type Class

Gonad

Cell type

Cumulus Cells

MeSH Description

The granulosa cells of the cumulus oophorus which surround the OVUM in the GRAAFIAN FOLLICLE. At OVULATION they are extruded with OVUM.

Sample

source_name

CC_input_rep1

tissue

cumulus cell

strain

BDF1

development stage

Terminal differentiation

strategy

input DNA

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

①SCNT procedure:Eight- to ten-week-old B6D2F1 (C57BL/6×DBA/2) female mice were superovulated by injection with 5 IU of pregnant mare serum gonadotropin (PMSG), followed by injection of 7 IU of human chorionic gonadotropin (hCG, San-Sheng Pharmaceutical) 48 h later. Mouse MII oocytes were retrieved from the dissected oviducts at 13 h post-hCG injection and incubated in Chatot-Ziomek-Bavister medium (CZB) at 37°C and 5% CO2 until use. CCs were removed by incubating the cumulus-oocyte complex in hyaluronidase from bovine testes (0.5 mg/ml, Sigma) at 37°C for 3-5 min. Fully dissociated cumulus cells were collected and washed in Hepes-CZB and then stored at 4°C until nuclear transfer manipulation. Mouse MII oocytes were enucleated in Hepes-CZB containing 5 μg/ml cytochalasin B (Sigma) at 37°C. The inner diameter of the transfer needles used for CC nucleus was 5-6 μm. The somatic cell was administered 1-2 piezo pulses (intensity 1 or 2) while at the tip of the transfer needle to break the plasma membrane. Then, the cell was immediately injected into an enucleated oocyte in Hepes-CZB containing 5 μg/ml cytochalasin B. The plasma membrane of the enucleated oocyte was broken with a single piezo pulse, the cumulus cell was injected, and the membrane was sealed by aspiration of a small amount of cytoplasm. Reconstructed embryos were cultured in CZB at 37°C and 5% CO2 for one hour and activated for 5 hours using strontium chloride in calcium-free CZB in the presence of 5 μg/ml cytochalasin B to prevent the extrusion of a pseudopolar body. The reconstructed embryos were then transplanted into G1 medium and cultured to the blastocyst stage. ②Sample harvest:embryos at each of the following stages were collected: 0.5 h and 1 h postinjection; 1 h, 6 h and 12 h postactivation; 2-cell, 4-cell, and 8-cell; morula; ICM and TE of day 3.5 blastocysts. An ES cell line (R1, male) and cumulus cells were also harvested for the Hi-C experiment. For the one-cell and cleavage-stage embryos, the zona pellucidae of the embryos were removed with 0.5% pronase E (Sigma), and the embryos were then incubated in calcium-free CZB for 5 min. Polar bodies were removed by gently pipetting using a firepolished glass needle with an inner diameter of 120 μm. For morula, ICM and TE isolation, the zona pellucidae of blastocysts were removed with 0.5% pronase E. The embryos were then incubated in calcium-free CZB for 20 min, and the tight junctions of morula, TE and ICM cells were separated by gently pipetting using a pipette with an inner diameter of 40–60 μm. ①Hi-C libaries:The generation of Hi-C libraries with a low number of cells was optimized according to a previous protocol. For Hi-C library generation and sequencing, 100 ~ 500 cells were used per reaction, and at least two replicates were analyzed for each stage. All isolated cells were washed three times with a 0.5% bovine serum albumin in phosphate-buffered saline (BSA-PBS, Sigma) solution to avoid potential contamination. DNA was sheared to 300 ~ 500 bp with the Covaris S220 instrument. Biotin-labeled DNA was then pulled down with 10 μl of Dynabeads MyOne Streptavidin T1 (Life Technologies, SA-T1). The sequencing library was prepared using beads with the KAPA hyper kit. In total, 14 ~ 16 cycles of PCR amplification were performed. DNA was removed from the SA-T1 beads by heating at 98°C for 10 min with an additional 15 μl of elution buffer. Finally, size selection was performed using AMPure XP beads. Fragments ranging in size from 200 bp to 500 bp were selected. All libraries were sequenced on the Illumina HiSeq X Ten or Nova platform. ②ULI-NChIP libaries:The ULI-NChIP libraries for CCs were generated according to a previous protocol45. At least 500 cells were used per reaction, and two replicates were performed. All isolated cells were washed three times with a 0.5% BSA-PBS solution to avoid potential contamination. One microgram of the histone H3K9me3 antibody (39161, Active Motif) was used for each immunoprecipitation reaction. Paired-end 150-bp sequencing was performed on the Illumina HiSeq X Ten platform. ③RNA-seq:ICM cells and TE cells were harvested as described above. For RNA-seq library construction and sequencing, 5 ~ 10 cells were used per reaction. All isolated cells were washed three times with 0.5% BSA-PBS solution to avoid potential contamination. A single-cell RNA-seq library was generated as described previously46. The RNA-seq libraries were generated using the KAPA Hyper Prep Kit according to the manufacturer's instructions. Single-end 50-bp sequencing was performed on the Illumina HiSeq 2500 platform.

Sequencing Platform

instrument_model

HiSeq X Ten

mm10

Number of total reads

36516380

Reads aligned (%)

81.6

Duplicates removed (%)

27.1

Number of peaks

161 (qval < 1E-05)

mm9

Number of total reads

36516380

Reads aligned (%)

81.5

Duplicates removed (%)

27.0

Number of peaks

139 (qval < 1E-05)