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| Status |
Public on Dec 08, 2019 |
| Title |
PGCLC_Par_IAA_r2 |
| Sample type |
SRA |
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| Source name |
Primordial germ cell-like cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Primordial germ cell-like cells
line: Parental
treatment: With auxin
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| Growth protocol |
PGC-competent 4i hESCs (WIS2-NANOS3-T2A-tdTomato) were cultured on irradiated mouse embryonic fibroblasts (MEFs) (GlobalStem) in 4i medium. Media were replaced every day. hESCs were passaged by single-cell dissociation using 0.25% Trypsin-EDTA (GIBCO). 10 μM ROCK inhibitor (Y-27632, TOCRIS) was added for 24 hours after passaging. To induce hPGCLCs, 4i hESCs were trypsinized, filtered and plated into ultra-low cell attachment U-bottom 96-well plates (Corning, 7007) at 4,000 cells/well density in 100 μl PGCLC medium. The plates were centrifuged at 300g for 3 minutes and placed into a 37 °C 5% CO2 incubator until embryoid body (EB) collection.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA-seq was performed on PRDM14-AID-Venus competent 4i hESCs and hPGCLCs induced therefrom. Two biological replicates were used for each condition. For IAA-sensitive cells, two clones (cl11 and cl21) from the same hESC passage or the same hPGCLC induction were used as replicates. For the parental (“no TIR1”) control, the same cell line was used at different passages or inductions to yield two independent replicates. 10,000 AP+ 4i hESCs or 10,000 NANOS3-tdTomato+AP+ hPGCLCs (with the exception of hPGCLC cl21 replicate, where 3,000 cells were used) were sorted directly into 100 μl of extraction buffer from PicoPure RNA Isolation Kit (Applied Biosystems) for subsequent total RNA extraction according to manufacturer’s protocol. RNA was stored at -80 ºC and its quality and quantity were checked by Agilent RNA 6000 Pico Kit with Bioanalyzer (Agilent Technologies) and Qubit (Thermo Fisher Scientific).
RNA-seq library was prepared from 10 ng input RNA using the end-to-end Trio RNA-seq library prep kit (Nugen) following the manufacturer’s protocol but omitting the AnyDeplete step. In short, the protocol contains the following steps: DNase treatment to remove DNA from RNA; first strand and second strand cDNA synthesis to produce the reverse complement of the input RNAs; cDNA purification using Agencourt AMPure XP beads (Backman Coulter); single primer isothermal amplification (SPIA) to stoichiometrically amplify cDNAs; enzymatic fragmentation and end repair; sequencing adaptor (index) ligation; product purification using AMPure beads; library amplification (4 cycles were used); library purification using AMPure beads. Libraries were then quantified by qPCR using NEBNExt Library Quant Kit (NEB) for Illumina on QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems). Fragment size distribution and the absence of adapter dimers was checked using Agilent TapeStation 2200 and High Sensitivity D1000 ScreenTape. Finally, RNA-seq libraries were subjected to single-end 50 bp sequencing on HiSeq 4000 sequencing system (Illumina). 24 indexed libraries (including samples from this work and 8 others) were multiplexed together and sequenced in two lanes of a flowcell, resulting in >30 million reads per sample.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
processed data file: DEseq2_log2_normalized_count.txt.gz
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| Data processing |
The library sequence quality in demultiplexed fastq files was checked by FastQC (v0.11.5) and the low-quality reads and adaptor sequences were removed by Trim Galore (v0.4.1) using the default parameters.
The pre-processed RNA-seq reads were mapped to the human reference genome (UCSC GRCh38/hg38) using STAR (2.6.0a) (--outFilterMismatchNoverLmax 0.05 --outMultimapperOrder Random --winAnchorMultimapNmax 100 --outFilterMultimapNmax 100) guided by the ENSEMBL (Release 87) gene models.
Read counts per gene were extracted using TEtranscripts and normalized by DEseq2 in R.
Genome_build: hg38 (GRCh38)
Supplementary_files_format_and_content: DEseq2_log2_normalized_count.txt.gz: Tab-delimited text file containing ensemble_gene_id, hgnc_symbol, biotype, gene coordinate and DEseq2 normalized expression level of each sample [log2(normalized counts +1)].
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| Submission date |
Oct 09, 2019 |
| Last update date |
Dec 08, 2019 |
| Contact name |
Walfred Tang |
| E-mail(s) |
walfredtang@gmail.com
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| Organization name |
University of Cambridge
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| Department |
Gurdon Institute
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| Lab |
Azim Surani
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| Street address |
Tennis Court Road
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| City |
Cambridge |
| ZIP/Postal code |
CB2 1QN |
| Country |
United Kingdom |
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| Platform ID |
GPL20301 |
| Series (2) |
| GSE138673 |
A critical but divergent role of PRDM14 in human primordial germ cell fate revealed by inducible degrons [RNA-seq] |
| GSE138675 |
A critical but divergent role of PRDM14 in human primordial germ cell fate revealed by inducible degrons |
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| Relations |
| BioSample |
SAMN13005838 |
| SRA |
SRX6973245 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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