Project description:Human oocyte cDNA library was hybridized on a multi-species oocyte array (Bovine, Mouse, Frog) Temperature stringency criteria was used to evaluate the conservation degree of oocyte genes among vertebrates (Bovine, Mouse, Frog)
Project description:Oocytes and granulosa cells were collected after natural and superovulation in young (3 month-old) and old (12 month-old) mice while conserving pairing information between oocyte and granulosa cells from each cumulus-oocyte-complex (COC). This experimental design allows for the analysis of the effects of ageing and / or superovulation on the transcriptome of COCs. The naming convention for samples is as follows: [ovulation, NO/SO][age, 3M/12M][cell, OC/GC][COC number], meaning that NO12MOC01 and NO12MGC01 are from the same COC. Oocyte and granulosa cell samples were processed for full-length cytoplasmic RNA amplification using slightly modified SMART-seq2 protocol. After amplification of cDNA and library preparation, samples were sequenced on NextSeq 550 using Single-Ended 75bp High-Output kit.
Project description:Human oocyte cDNA library was hybridized on a multi-species oocyte array (Bovine, Mouse, Frog) Temperature stringency criteria was used to evaluate the conservation degree of oocyte genes among vertebrates (Bovine, Mouse, Frog) 2 technical replicates on distinct array were made at each pre-determined hybridization temperature (45°C, 50°C, 55°C), overall, the experiment includes 6 arrays
Project description:Considerable effort has been devoted to refining experimental protocols having reduced levels of technical variability and artifacts in single-cell RNA-sequencing data (scRNA-seq). We here present evidence that equalizing the concentration of cDNA libraries prior to pooling, a step not consistently performed in single-cell experiments, improves gene detection rates, enhances biological signals, and reduces technical artifacts in scRNA-seq data. In this submission the original single-cell cDNA is from GSE75748 cells labelled EC2 and TB2 in which the cDNA was not equalized prior to library preparation. We performed a set of experiments with the equalization step and provide three count matrices - 1) EQ (equalized cDNA prior to pooling in equal amounts); 2) EQ Vary (same as EQ but pooled at unequal amounts); 3) EQ-75% (equalized cDNA and pooled equally, sequeced to lower total depth).
Project description:Purpose: The goal of this study is to analyze microRNA affected by wuho and mei-p26 mutations in Drosophila ovary Ovarian microRNA prolife of 7 day old control flies and flies bearing wuho and mei-p26 mutations, in duplicate.Sample libraries were prepared using the QIAseq miRNA Library Kit (QIAGEN) according to the manufacturer's protocols. Adaptors are ligated sequentially to the 3' and 5' ends of miRNAs. Subsequently, universal cDNA synthesis with UMI assignment, cDNA cleanup, library amplification and library cleanup are performed. Libraries were sequenced on an Illumina instrument (75-cycle single-end read, 75SE). Sequencing data was processed using the Illumina software BCL2FASTQ v2.20.
Project description:In this study we describe a new method for GTO (Genomics Transcriptomics One-Tube) that allows for simeltaneous sequencing of RNA and DNA from a single cell in the same tube. This method relies on dual amplification. The first amplification is done immediately after the mRNA is transcribed to cDNA using SMART-Seq2 protocol (Picelli et al., 2014) to increase the level of cDNA to that comparable to gDNA. The second amplification with universal primers from SeqXE kit for whole genome amplification (from Sigma Aldrich) amplifies the cDNA and gDNA fragments to amounts required for library preparation. The reads generated from this mixed library are separated bioinformatically to exonic and nonexonic reads depending on where they align in the reference genome to generate the RNAseq and DNAseq data respectively. We present data generated from single in-vitro cells of multiple cell lines (A375, BT549, SKBR3, 315A) and also from single in-vivo cells isolated from tumors of an orthotopic tranplantation mouse model of pancreatic cancer (using KPC1199-EGFP cell line).
