GSM2483568: S5_mRNA_seq; Mus musculus; RNA-Seq
Sample collection and preparation. Metaphase II (MII) oocytes were collected from superovulated sexually mature female Kunming mice. The mouse oviducts were dissected and placed in M2 media, and extracted the cumulus cell complex. Then we removed the cumulus cells around the oocytes by treatment with hyaluronidase (sigma), washed the oocytes by pipetting them 4-6 times with M2 media, and collected the oocytes under a stereomicroscope by mouth pipetting. The oocytes were treated by microinjection system (Eppendorf). Next we transferred the oocytes to a drop of M2 media under mineral oil (Sigma) in a 3.5-cm dish, partially removed zone pellucida by laser-assisted biopsy, and collected the first body and nucleus by a micropipette in 0.2 mL PCR tube with 5 μL Nuclease-free distilled water (InvitrogenTM), respectively. Then we collected cytoplasm (oocytes without nucleus or first body) in a 0.2 mL PCR tube with 5 μL lysis buffer. The nucleus, first bodies and cytoplasm were stored at -80℃ until required for library preparation. Negative controls were either Nuclease-free water or lysis buffer alone. Single cell DNA amplification. We amplified the genomic DNA from single isolated nucleus. Out of six different samples, two samples were amplified by REPLI-g Single Cell Kit (Qiagen, Cat no.150345) based on multiple displacement amplification (MDA) method. In short, single nucleus was lysed and denatured at 65oC for 10 minutes, then the DNA amplification was starting with the random hexamer primers binding to the template in the reaction tube by a high fidelity f29 DNA polymerase and incubating at 30oC for 8 hours. The reactions were then inactivated at 65oC for 3 minutes and stored in -80oC for holding. The other four samples were amplified by GenomePlex single cell amplification kit (Sigma-Aldrich, Cat no. WGA4-50RXN). In short, we first lysed the nucleus and digested the proteins by protease K at 50 °C for 1 hour. The genomic DNA was fragmented into 200-400bp at 99 °C for 4 minutes . Random primers linked with common adaptors were annealed to the fragmented DNA template using the following incubation setting: 16 °C, 20 minutes, 24 °C, 20 minutes, 37 °C, 20 minutes, 75 °C, 5 minutes, and 4 °C hold. Then, the mixture were amplified with an initial denaturation at 95 °C, 3 minutes, and 25 cycles of 94 °C, 30 seconds and 65 °C, 5 minutes. The amplified products were purified using the Qiagen PCR purification reagents. The sequencing libraries were constructed by BGI-Shenzhen and sequenced using the Illumina HiSeq 2000 sequencing platform. Exome-seq. We used SureSelectQXT exome enrichment kit (Agilent technologies Inc., Germany) to capture and enrich the exome regions from the sequencing library of single cell whole genomic DNA. In short, we mixed the sequencing library from single nucleus with SureSelect exome probes, which were tagged with magnetic labelling, and incubated at 65oC for 24 hours (optimal, 72hours), allowing the probe hybridized to the library thoroughly. Then the purified sequences were further amplified by PCR and purified. After quantity assessment by bioanalyzer, they were sequenced on the illumina HiSeq 2000 sequencing platform (illumina co., CA, USA). Single cell RNA amplification. Single cell transcriptome of the enucleated cytoplasm was amplified by SMARTer ultra low RNA kit (Clontech, Cat. no. 634936) as the manual instructed. Briefly, we first synthesized the first strand cDNA from single enucleated cytoplasm using modified oligo(dT) (SMART CDS primer) and then tailed several additional nucleotides to the 3’end of the first strand cDNA due to the enzyme’s terminal transferases activity. Common adaptor (SMARTer II A oligonucleotide) were linked to the 3’end of the first strand cDNA. The resulting full-length single strand cDNAs started with poly T and ended with a common adaptor. Then full transcriptome of the enucleated cytoplasm was amplified by universal primers (by common adaptor and oligod(T) ) and ready for sequencing library construction. The sequencing libraries were constructed by BGI-Shenzhen and sequenced using the Illumina HiSeq 2000 sequencing platform.
GEO Accession
GSM2483568