 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 19, 2022 |
| Title |
4D diff G2 1 in bulk |
| Sample type |
SRA |
| |
|
| Source name |
R1 mESC cell line
|
| Organism |
Mus musculus |
| Characteristics |
tissue: R1 mESC cell line
developmental stage: Embrionic stem cells in early differentiation
state: RA differentiation
differentiated days: 4
|
| Treatment protocol |
RA (Sigma-Aldrich, R2625) dissolved in dimethyl sulfoxide (ENCO, 0219605580) at 1 μM in LIF-free media with 10% FBS, on 0.1% gelatin-coated plates.
|
| Growth protocol |
ESCs were seeded on 0.1% gelatin-coated plates (Sigma-Aldrich, G1393) and grown in ES medium (Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, D5671), 10% fetal bovine serum (FBS, Biological Industries, 04-007-1A), 1 mM sodium pyruvate (Biological Industries, 03-042-1B), 0.1 mM nonessential amino acids (Biological Industries, 01-340-1B), 0.1 mM β-mercaptoethanol (Sigma-Aldrich, M3148), 1000 U/ml LIF (Mercury, ESG1107)). The cells were maintained at an incubator at 37 °C with 5% CO2 humidified air. For pluripotent conditions, 3 µM CHIR99021 (PeproTech, SM-2520691-B) and 0.2 µM PD0325901 (PeproTech, SM-3911091-B) were added to the ES medium.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
mRNA extraction was performed using Invitrogen Dynabeads mRNA DIRECT Purification Kit (61011) according to the protocol included with the kit.
The library for differential gene expression analysis was prepared using 100-1000 ng of mRNA. First, RNA was fragmented using an RNA fragmentation kit (Ambion, AM8740) following the manufacturer’s protocols. Fragmented RNA was purified with 1.4x reaction volume of AMPure XP beads (Beckman Coulter, A63881) and eluted in 10 μl TE buffer (10 mM Tris–HCl, pH 8.0, 0.1 mM EDTA). The resulting fragmented RNA was reverse transcribed using an oligo dT primer: First, 10 μl of RNA were mixed with 2 μl of 50 μM oligo dT primer (Bio-Rad, 1725038) and 1 μl of 25 mM dNTP mix (NEB, N0447S), and samples were incubated for 3 minutes at 65 °C and transferred to ice. The following components were added to the reaction, for a total volume of 20 μl: 4 μl of 5x First-Strand SuperScript III buffer (Invitrogen, 18080044), 2 μl of 0.1 M DTT (Invitrogen, 18080044), 1 μl murine RNase inhibitor (NEB, M0314L), and 1 μl of 200 U/μL SuperScript III RT enzyme (Invitrogen, 18080044). The reaction product (in the form of a cDNA:RNA hybrid) was purified with 1.5x reaction volume of AMPure XP beads (Beckman Coulter, A63881) and eluted in 16 μl TE buffer as described above. For second-strand synthesis, 16 μl of the digestion reaction product were combined with 2 μl second-strand synthesis (SSS) buffer and 1 μl of SSS enzyme mix of mRNA Second Strand Synthesis Module kit (NEBNext, E6111S) and incubated at 16 °C for 2.5 hours, followed by 20 minutes at 65 °C. The resulting product was purified with 1.5x reaction volume of AMPure XP beads (Beckman Coulter, A63881) and eluted with 20.4 μl TE buffer. cDNA edges were repaired using End-It DNA End-Repair Kit (Danyel Biotech, ER81050) by combining the 20.4 μl sample with 3 μl 10X End-repair Buffer, 3 μl 2.5 mM dNTPs, 3 μl 10 mM ATP, and 0.6 μl END-IT enzyme mix at room temperature for 45 minutes, followed by purification with 1.5x reaction volume of AMPure XP beads (Beckman Coulter, A63881) and elution with 32 μl TE buffer. Klenow-mediated addition of an adenine to the 3’ end of the DNA fragments was performed using the Klenow fragment (3'→5' exo-) kit (NEB, M0212L) by combining the 32 μl sample with 5 μl 10X Klenow Buffer NEB 2, 10 μl 1 mM dATP, and 0.6 μl 5 U/μl Klenow (3’-5’ exo-) at 37 °C for 30 minutes, followed by purification with 1.5x reaction volume of AMPure XP beads (Beckman Coulter, A63881) and elution with 8 μl TE buffer. Illumina library adaptors were added to the resulting cDNA fragment using DNA ligase (NEB, M2200S) by incubation with 5 μl 2x Ligase Buffer, 0.5 μl adapter Oligo mix (1:10 in H2O), and 0.5 μl 1 U/ml DNA Ligase at room temperature for 15 minutes. The ligated product was purified with 1.5x reaction volume of AMPure XP beads (Beckman Coulter, A63881) and eluted in 11.5 μl TE buffer. The resulting libraries were PCR amplified using standard PE1/PE2 full-length primer mix containing Illumina library indices for multiplexing (sequences are given in Supplementary Table X). Each PCR reaction contained 11.5 μl post-RT cDNA library, 12.5 μl 2x KAPA HiFi HotStart PCR mix (Zotal, KK-KK2601), and 1 μl of 25 μM 2p Fixed (+barcode) and 2p Fixed primer mix (Supplementary Table 3). Amplified libraries were purified using 0.7x reaction volume of AMPure XP beads (Beckman Coulter, A63881) and eluted in 32 μl TE buffer. A 15-μl aliquot of each resulting library was run in a 2% agarose gel, and size selection for the desired 200-800 bp DNA library fragments was performed using PureLink DNA gel extraction kit (Invitrogen, K210012). Library quality was confirmed by Agilent 2200 TapeStation Nucleic Acids System. The in vitro transcription sequencing (IVT) library was prepared using 75-100 ng of mRNA, extracted as described above. First-strand synthesis was performed using SuperScript III Reverse Transcriptase (Invitrogen, 18080044) and the T7RTPolyT primer (Supplementary Table X) at 50 °C for 2 hours followed by 15 minutes at 70 °C and 1 minute at 4 °C. To remove unused primers and primer dimers, the reaction product was combined with 20 μl digestion mix containing 5 μl ExoI (NEB, M0293S), 5 μl HinFI (NEB, R0155S), 5 μl ExoI buffer (NEB, B0293S), 5 μl CutSmart buffer (NEB, B7204S), and 30 μl nuclease-free water and incubated for 1 hour at 37 °C and 10 minutes at 80 °C. The reaction product (in the form of a cDNA:RNA hybrid) was purified with 1.5x reaction volume of AMPure XP beads (Beckman Coulter, A63881) and eluted in 13.5 μl TE buffer. For second-strand synthesis, the 13.5 μl digestion reaction product was combined with 1.5 μl SSS buffer and 1 μl of SSS enzyme mix from the mRNA Second Strand Synthesis Module kit (NEBNext, E6111S) and incubated at 16 °C for 2.5 hours, followed by 20 minutes at 65 °C. For linear amplification by in vitro transcription, 16 μl of SSS reaction products were combined with 24 μl HiScribe T7 High Yield RNA Synthesis Kit (NEB, E2040S) reagent mix containing 4 μl T7 Buffer, 4 μl ATP, 4 μl CTP, 4 μl GTP, 4 μl UTP, and 4 μl T7 enzyme mix. The reaction was incubated at 37 °C for 13 hours, and the resulting RNA was purified with 1.3x reaction volume of AMPure XP beads (Beckman Coulter, A63881) and eluted with 20 μl TE buffer. Aliquots of 9 μl of each sample were frozen for backup at -80 °C, 2 μl of each sample was directly analyzed, and the remaining 9 μl were used in subsequent library preparation steps. RNA was fragmented using the RNA fragmentation kit (Ambion, AM8740) by combining 9 μl sample with 1 μl of RNA fragmentation reagent and incubating at 70 °C for 2 minutes. After transfer to ice, 40 μl fragmentation stop mix containing 5 μl fragmentation stop solution and 35 μl TE buffer was added. Fragmented RNA was purified with 1.4x reaction volume of AMPure XP beads (Beckman Coulter, A63881) and eluted in 10 μl TE buffer. The resulting amplified and fragmented RNA was reverse transcribed using a random hexamer primer (IDT) as follows: First, 10 μl of RNA were mixed with 2 μl of 100 μM PvG748-SBS12-RT random hexamer primer (Supplementary Table X) and 1 μl of 10 mM dNTP mix (NEB, N0447S), incubated for 3 minutes at 65 °C and transferred to ice. The following components were then added to the reaction for a total volume of 20 μl: 4 μl of 5x First-Strand SuperScript III buffer (Invitrogen, 18080044), 1 μl 0.1 M DTT (Invitrogen, 18080044), 1 μl murine RNase inhibitor (NEB, M0314L), and 1 μl of 200 U/µL SuperScript III RT enzyme (Invitrogen, 18080044). Following reverse transcription, the reaction volume was raised to 50 μl by adding 30 μl nuclease-free water, and the resulting cDNA was purified with 1.2x reaction volume of AMPure XP beads (Beckman Coulter, A63881) and eluted in 11.5 μl TE buffer. Library amplification was performed using 12.5 µl 2x KAPA HiFi HotStart ReadyMix PCR kit (Zotal, KK-KK2601) and 1 μl of 25 μM 2p fixed primers (Supplementary Table X). Amplified libraries were purified using 0.7x reaction volume of AMPure XP beads (Beckman Coulter, A63881) and eluted in 30 μl nuclease-free water. Aliquots of 15 μl of each library were run in 2% agarose gels, and size selection for the desired 200-600 bp DNA library fragments was performed using PureLink DNA gel extraction kit (Invitrogen, K210012). Library quality was confirmed using the Agilent 2200 TapeStation Nucleic Acids System (Agilent). For scRNA-seq, encapsulation of cells with Reverse Transcription mix (IGEPAL CA-630- Sigma-Aldrich I8896-50ML , SuperScript III Reverse Transcriptase Invitrogen 18080044, Deoxynucleotide (dNTP) Solution Set Ornat N0446S, D,L-DITHIOTHREITOL Bio-Lab 000448235200, TRIS-HCL 1 M STOCK SOLUTIONS PH 7.5 B 1L Sigma-Aldrich T2319-1L, RNase Inhibitor, Murine NEB M0314L) and gels with unique molecular identifiers was performed using the FLUIGENT Smart Microfluidics Pump Syste
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Illumina output was analyzed using an in-house Perl script that produced a reads matrix that was aligned using RSEM with Bowtie. The resulting matrix was analyzed in R
For bulk data analysis the transcript per million (TPM) values were used to compare between libraries
scRNA-seq data was analyzed using the Seurat v2.4 pipeline
cells with more than 10,000 unique molecular identifiers were retained for further analysis. A global-scaling normalization was performed on the filtered dataset using “LogNormalize” with a scale factor of 10,000. Identification of highly variable genes was performed with the following parameters: x.low.cutoff = 0.2, x.high.cutoff = 5, y.cutoff = 0.5, and y.high.cutoff = 10. Cell-to-cell variation in gene expression driven by batch, cell alignment rate, and the number of detected molecules were regressed out and a linear transformation was applied. A principal component analysis was performed on the scaled data with 12 principal components. Clustering was done with resolution of 0.6, and tSNE or UMAP was using for visualization.
CHIP-seq Sequencing data were aligned using Bismark and Bowtie (https://www.bioinformatics.babraham.ac.uk/projects/bismark/Bismark_User_Guide.pdf) using paired-ended approach. TDF genomic browser files were produced using IGV count. We applied HOMER to find peaks using ChIP-seq criteria and used BEDTools to intersect bins with genomic intervals such as promoters, genes, and predicted enhancers.
Genome_build: mm9, GRC38
|
| |
|
| Submission date |
Jun 17, 2021 |
| Last update date |
May 20, 2022 |
| Contact name |
Muhammad Awawdy |
| E-mail(s) |
muhammad.awawdy@mail.huji.ac.il
|
| Phone |
+972528482707
|
| Organization name |
Hebrew university of jerusalem
|
| Lab |
Muhammad Awawdy
|
| Street address |
The Hebrew University, The Edmond J. Safra Campus - Givat Ram
|
| City |
jerusalem |
| ZIP/Postal code |
9190401 |
| Country |
Israel |
| |
|
| Platform ID |
GPL19057 |
| Series (1) |
| GSE178390 |
Cell cycle states of pluripotent cells dictate lineage decisions |
|
| Relations |
| BioSample |
SAMN19762092 |
| SRA |
SRX11172317 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5389579_d4G2_1_rsem_mm9_output.genes.results.gz |
656.5 Kb |
(ftp)(http) |
RESULTS |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
