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Sample GSM2635793

Query DataSets for GSM2635793

Status

Public on Mar 14, 2018

Title

E14.9_p3

Sample type

SRA

Source name

Day2_8-cell stage

Organism

Bos taurus

Characteristics

developemental stage: 8-cell stage
embryo source: E2-Day2
cell barcode: ATTCTA

Treatment protocol

None

Growth protocol

Embryos were produced in vitro according to a standard procedure including in vitro maturation (IVM) and fertilization (IVF) (Reichenbach et al., 2010, Transgenic Research 19, 549-556). Briefly, follicles from slaughterhouse ovaries were aspirated and obtained cumulus-oocyte- complexes (COCs) matured for 23 h in MPM supplemented with LH and FSH and 5% estrous cow serum (ECS). Matured COCs were co-incubated with frozen-thawed sperm, prepared by the swim-up method. For IVM and IVF, COCs were incubated at 39°C in a maximum humidified atmosphere of 5% CO 2 in air. After 20 h of co-incubation, presumptive zygotes were vortexed to remove remaining cumulus cells and transferred to synthetic Oviductal fluid (SOF) supplemented with 5% ECS, 400 μl BME, 100 μl MEM under mineral oil and cultured at 5% CO 2 , 5% O 2 , 90% N 2 and 39°C in humidified air.

Extracted molecule

total RNA

Extraction protocol

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.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 1500

Data processing

SCRB-Seq libraries were demultiplexed from Nextera barcodes and cell barcodes
Alignments were calculated using STAR 2.5.2b using all default Parameters.
UMI tables were generated using the published Drop-seq pipeline (Macosko et al., 2015. Cell 161, 1202-1214)
Genome_build: Bos tau7 (NCBI) and ERCC spike-in reference
Supplementary_files_format_and_content: The 'umi_bosTau_ERCC.txt' contains the endogenous and ERCC UMI counts. These count has been performed after running Drop-seqAll reads were mapped to Bos tau7 (NCBI) and ERCC spike-in reference. Alignments were calculated using STAR 2.5.2b using all default parameters. UMI tables were generated using the published Drop-seq pipeline.
Supplementary_files_format_and_content: The normalized_UMI.txt: Data were then normalized in the following way, to account for differences in efficiency of transcript recovery between wells: UMI counts were divided by the total number of UMI counts per cell and then multiplied by the median of total UMI counts across all cells.

Submission date

May 23, 2017

Last update date

May 15, 2019

Contact name

Ilaria Lavagi

E-mail(s)

ilaria.lavagi@gmail.com

Organization name

Ludwid Maximilian University

Street address

Feodor Lynen Strasse 25