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Sample GSM3160739

Query DataSets for GSM3160739

Status

Public on Jun 24, 2018

Title

scRNA-Seq_SIII D3

Sample type

SRA

Source name

Intermediate cells on stage III day 3 during chemical reprogramming of MEFs

Organism

Mus musculus

Characteristics

cell type: Intermediate cells
strain: C57BL/6J-Tg(GOFGFP)11Imeg/RbrcxICR
time: stage III day 3

Treatment protocol

MEFs were seeded at a density of 50,000 cells per well of a 6-well plate or 300,000 cells per 100 mm dish with MEF culture medium. The next day (day 0), change the medium into stage I medium. Change the medium every 4 days. On day 12, for the induction with FBS/KSR-SII condition, re-plate the cells at a density of 50,000-200,000 cells per well of a 6-well plate with stage I medium reduced concentrations of bFGF and CHIR99021). For the induction with N2B27-SII condition, feeder cells (MEFs treated with mitomycin C and seeded at 150,000 cells per well of 6-well plate) should be prepared one day before re-plating. Re-plate the cells on feeder cells at a density of 2,000-10,000 cells per well of 6-well plate with stage I medium (reduced concentrations of bFGF and CHIR99021). On day 16, XEN-like epithelial colonies are formed and the culture is changed into stage II medium. On day 28, change the culture into stage III medium.

Growth protocol

MEFs were cultured in high glucose DMEM (Gibco) supplemented with 15% fetal bovine serum (FBS, Pan-Biotech), 1% GlutaMAX-I (Gibco), 1% nonessential amino acids (NEAA, Gibco), 0.1 mM β-mercaptoethanol (Sigma-Aldrich) and 1% penicillin-streptomycin (Gibco). Mouse ESCs and CiPSCs were maintained on feeder layers (the mitomycin C-treated MEFs) in ESC culture medium containing KnockOut DMEM (Gibco), 10% knockout serum replacement (KSR, Gibco), 10% FBS, 1% GlutaMAX-I, 1% NEAA, 0.1 mM β-mercaptoethanol, 1% penicillin-treptomycin supplemented with 2iL (3 µM CHIR99021, 1 µM PD0325901 and 10 ng/ml LIF) or with additional 50 μg/ml vitamin C (for scRNA-seq in Figure 6A). MEFs, ESCs and CiPSCs were incubated at 37°C with 5% CO2.

Extracted molecule

polyA RNA

Extraction protocol

Total RNA was isolated using Direct-zol RNA MiniPrep Kit (Zymo Research). Genomic DNA of sorted pZscan4c-Em+, pZscan4c-Em- cells at stage II D12, XEN-like cell with pZscan4c-Emerald reporter and CiPSCs were extracted using EasyPure Genomic DNA Kit (TransGen, EE101-11).
Total RNA was isolated using Direct-zol RNA MiniPrep Kit (Zymo Research). RNA sequencing libraries were constructed using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB England BioLabs).. Genomic DNA of sorted pZscan4c-Em+, pZscan4c-Em- cells at stage II D12 and pZscan4c-Emerald XEN-like cell were extracted using EasyPure Genomic DNA Kit (TransGen, EE101-11). DNA were denatured and bisulfite converted using EZ DNA Methylation-GoldTM Kit (Zymo Research, D5005). In brief, 20 µl DNA (300 ng) was mixed with 130 µl CT Conversion Reagent and performming the following steps: 98°C for 10 minutes, 64°C for 2.5 hours. Samples and 600 µl M-Binding Buffer were added into a Zymo-Spin™ IC Column, followed by inverting the column several times and centrifuging at full speed (>10,000 x g) for 30 seconds. 100 µl M-Wash Buffer was added to the column and centrifuged at full speed for 30 seconds. Add 200 µl M-Desulphonation Buffer to the column, incubate at room temperature (20-30°C) for 15-20 minutes and then centrifuging at full speed for 30 seconds. Wash the column with 200 µl M-Wash Buffer twice and elute the DNA with 10 µl M-Elution Buffer. Recovered bisulfite-converted DNAs were constructed into sequencing libraries using Accel-NGS Methyl-Seq DNA Library Kit (Swift, 30024) following the manufacturer’s instructions. Cells at different timepoints throughout chemical reprogramming process were harvested and resuspended at 1 × 106 cells per milliliter in 1 × PBS with 0.04% BSA. Then, cell suspensions (300-1000 living cells per microliter determined by trypan blue staining) were loaded on a Chromium Single Cell Controller (10x Genomics) to generate single-cell gel beads in emulsion (GEMs) by using Single Cell 3' Library and Gel Bead Kit V2 (10x Genomics, 120237). Captured cells were lysed and the released RNA were barcoded through reverse transcription in individual GEMs (Zheng et al., 2017). Barcoded cDNAs were pooled and cleanup by using DynaBeads® MyOne™ Silane Beads (Invitrogen, 37002D). Single-cell RNA-seq libraries were prepared using Single Cell 3' Library Gel Bead Kit V2 (10x Genomics, 120237) following the manufacture’s introduction.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

HiSeq X Ten

Description

AGG.tar

Data processing

scRNA-Seq: We checked the quality of raw sequencing data by FastQC software. The read2 fastq sequence was trimmed by FASTX-Toolkit. We used 10x genomics cellranger count pipeline 2.0.1 to analyze the sequencing data and generated the single cell information, once for each individual sample. All the cells in different batches were merged together by cellranger aggr pipeline and normalized by equalizing the read depth among libraries. The final result was the matrix of all cells and their global gene expressions.
WGBS: WGBS analysis was performed using Bismark/Bowtie2 algorithm. The clean fastq format reads were obtained from the sequencer and aligned to mouse mm10 reference with Bismark in directional mode using the default parameters. The deduplication tool from Bismark was used to remove duplicate reads. The methylation calls were extracted from the unique read alignment using bismark_methylation_extractor and bismark2bedGraph command. The methylation calls from both Watson and Crick strand were combined for the following analysis.
Genome_build: mm10
Supplementary_files_format_and_content: scRNA-Seq: UMI counts matrix with gene and barcode annotations (where rows are genes, and columns are cell barcodes); RNA-Seq: FPKM matrix; WGBS: bedGraph

Submission date

May 27, 2018

Last update date

Jun 24, 2018

Contact name

Yifang Liu

E-mail(s)

liuyifang3000@gmail.com

Organization name

Peking University

Street address

Haidian District