Illumina HiSeq 2000 sequencing; GSM2635778: E1.1_p3; Bos taurus; RNA-Seq

E1.1_p3

we analysed the transcriptome of bovine embryo during the major Embryonic Gene Activation (EGA) in cows. We did it at a single cell level to investigate if the cell are heterogeneous and to check if early specification events take place already at this stage. Overall design: In vitro fertilization of bovine embryos. Single cell RNA sequencing at 8- and 16-cell stages. SCRB-seq library Please note that [1] sample titles represent embryo, blastomere and plate number. For example,  E14.1_p3 means exactly embryo 14, blastomere 1, plate 3. [2] more than one cell has origin from the same embryo. For example, E2-Day2 (in the source embryo) means Embryo 2 collected at Day 2.

scRNA-seq 8- to 16-cell stage bovine embryo

Submitted by Gene Expression Omnibus on 16-MAR-2018

The zona pellucida was removed by treatment with 5 mg/ml pronase for 1 min. nzyme reaction was stopped by washing embryos in THP supplemented with 10% fetal calf serum (FCS) and dissolved ZP was completely removed by gentle pipetting. Embryos were incubated in drops of PBS without Mg 2+ and Cl 2+ supplemented with 4 mg/ml polyvinylpyrrolidone under oil for 5-10 min and blastomeres were subsequently disaggregated by gentle pipetting (Simmet et al., 2015). Single cells were transferred individually to 0.5 μl drops of Buffer A under mineral oil (Prelude Direct Lysis Module, NuGEN), collected in a 384-well plate and stored at -80 °C Single Cell RNA Barcoding and Sequencing (SCRB-Seq) libraries were prepared (Soumillon et al., 2014). Briefly, the plate was thawed and 0.1 μl (1:10 6) ERCC RNA Spike-In Mix (Life Technologies) was added to each well; the addition of exogenous molecules in equal amount enabled us to later perform quality control. Cell barcode are reported in section “samples”. Subsequently, the construct, which contains cell barcodes (6 nt), the Unique Molecular Identifiers (UMIs; 10 nt) and a polyT with the anchor, was incorporated to the sequence through the template switching reaction while RNA was reverse transcribed; Maxima H Minus Reverse Transcriptase (Thermo Scientific) was used. After this step, the samples were pooled. Not-incorporated barcode primers were removed through digestion with Exonuclease I (New England Biolabs). Full-length cDNA amplification was performed with the KAPA HiFi HotStart polymerase (KAPA Biosystems). Nextera XT libraries were constructed from 1 ng of pre-amplified cDNA with a custom P5 primer (IDT). The libraries were sequenced paired-end with 16 cycles to decode cell barcodes and UMI from read 1 and 45 cycles into the cDNA fragment.

scRNA-seq 8- to 16-cell stage bovine embryo

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Samples