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Sample GSM6033298 Query DataSets for GSM6033298
Status Public on Apr 19, 2022
Title E6_MII_BTG4_KO_rep2_paiso
Sample type SRA
Source name Oocyte
Organism Mus musculus
Characteristics genotype: Btg4 KO
developmental stage: MII oocyte
molecule: Poly(A) tail length
Extracted molecule total RNA
Extraction protocol To obtain oocytes or preimplantation embryos, 8-weeks-old or 5-weeks-old C57BL/6 female mice were intraperitoneally injected with pregnant mare’s serum gonadotropin (PMSG; 5 IU) and human chorionic gonadotrophin (hCG; 5 IU). Preimplantation embryos were collected from 5-weeks-old C57BL/46 female mice mated with PWK/PhJ male mice.
Total RNA was extracted by TRIzol (life tech; 15596018) and further purified through isopropanal precipitation and resolved in 11 μl nuclease-free water. Total RNA is used for end extension with dU-containing end extension oligos by Klenow fragment (3'→ 5' exo-, NEB; M0212L). The end-extended RNAs (>200 nt) was cleaned up by RNA Clean & Concentrator-5 kit (Zymo; R1015) and eluted by 7 μl nuclease-free water. The cleaned RNA was reverse transcribed by SuperScript II similar to Smart-seq2 method. The amplified DNA was size selected by Ampure beads and made into SMRTbell Template library. The libraries were sequenced by Pacbio sequel II.
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Sequel II
Description E6_polyAtaillength_merge.txt
Data processing Ribo-lite reads were trimmed with cutadapt v1.14 with parameters: cutadapt --trim-n -a GATCGGAAGAGCACACGTCTG -a AGAGCACACGTCTG <input.file> | cutadapt -u 3 -a A{100} --no-indels -e 0.16666666666666666 - | cutadapt -O 8 --match-read-wildcards -g GTTCAGAGTTCTACAGTCCGACGATCSSS -m 18 - -o <output.file> , and the trimmed reads were sequentially mapped to mouse/human rRNAs sequences (mm9/hg19) using Bowtie2 v2.2.2 with parameters --seedlen=23. Those aligned to rRNA were discarded, and the rest reads were mapped to transcriptome of mm9/hg19 using STAR v2.5.3a with parameters --outFilterMismatchNmax 2 --outFilterMultimapNmax 20 --outFilterMatchNmin 16 --alignEndsType EndToEnd. The gene expression level was then calculated by Cufflinks v2.2.1 based on the annotation of CDS region。
All mRNA-seq data were trimmed by Trim Galore v0.4.2 then mapped to transcriptome of mm9/hg19 by STAR v2.5.3a with parameters --outFilterMultimapNmax 20 --outSAMstrandField intronMotif. The gene expression level was calculated by Cufflinks v2.2.1.
After sequencing, CCS reads were extracted. The CCS reads were further split into different samples based on barcode and trimmed into clean reads. (The uploaded data are splitted and trimmed already). The clean reads were aligned to the mm9 genome using minimap. The poly(A) tail length of each transcript was calculated by the length of the clipped sequence. The poly(A) tail length of a gene was presented by the genomic mean of the poly(A) tail length of all the transcript as described44. For MII poly(A) tail length analysis, we merged WT and Btg4+/- data as MII control poly(A) tail length data to increase data depth after confirming their similarity.
Assembly: mm9
Library strategy: PAIso-seq
Submission date Apr 07, 2022
Last update date Apr 19, 2022
Contact name Zhuqing Xiong
Organization name School of Life Science, Tsinghua Univers
Street address Haidian District
City Beijing
State/province FOREIGN
ZIP/Postal code 100084
Country China
Platform ID GPL29177
Series (1)
GSE165782 Ultrasensitive Ribo-seq reveals translational landscapes during mammalian oocyte-to-embryo transition and pre-implantation development
BioSample SAMN27404722
SRA SRX14778622

Supplementary data files not provided
SRA Run SelectorHelp
Processed data are available on Series record
Raw data are available in SRA

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