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Sample GSM3388564

Query DataSets for GSM3388564

Status

Public on Feb 20, 2019

Title

sci3-me-089

Sample type

SRA

Source name

mouse embryos

Organism

Mus musculus

Characteristics

developmental stage: mouse embryo (E9.5, E10.5, E11.5, E12.5, E13.5)
genetic background: B6 background

Growth protocol

The C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and plug matings were set up. Noon on the day of the vaginal plug was considered as embryonic day (E) 0.5. Dissections were done as previously described76 and all embryos were immediately snap frozen in liquid nitrogen. All animal procedures were in accordance with institutional, state, and government regulations (IACUC protocol 4378-01).

Extracted molecule

polyA RNA

Extraction protocol

Mouse embryos from different development stages were processed together to reduce batch effect. Each mouse embryo was minced into small pieces by blade in 1 mL ice-cold cell lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% IGEPAL CA-630 from77, modified to also include 1% SUPERase In and 1% BSA) and transferred to the top of a 40um cell strainer (Falcon). Tissues were homogenized with the rubber tip of a syringe plunger (5ml, BD) in 4ml cell lysis buffer. The filtered nuclei were then transferred to a new 15ml tube (Falcon) and pelleted by centrifuge at 500xg for 5min and washed once with 1ml cell lysis buffer. The nuclei were fixed in 4ml ice cold 4% paraformaldehyde (EMS) for 15min on ice. After fixation, the nuclei were washed twice in 1ml nuclei wash buffer (cell lysis buffer without IGEPAL), and re-suspended in 500ul nuclei wash buffer. The samples were split to two tubes with 250ul in each tube and flash frozen in liquid nitrogen. We estimated the nuclei extraction efficiency based on extracted nuclei number vs. expected total nuclei number in each embryo and the nuclei extraction efficiency range from 60% to 85%.
Thawed nuclei are permeabilized with 0.2% tritonX-100 (in nuclei wash buffer) for 3 minutes on ice, and briefly sonicated (Diagenode, 12s on low power mode) to reduce nuclei clumping. The nuclei were then washed once with nuclei wash buffer and filtered through 1ml Flowmi cell strainer (Flowmi). Filtered nuclei were spun down at 500xg for 5min and resuspended in nuclei wash buffer. Nuclei from each mouse embryo were then distributed into several individual wells in four 96-well plates. The links between well id and mouse embryo were recorded for downstream data processing. For each well, 80,000 nuclei (16 µL) were mixed with 8 µl of 25 µM anchored oligo-dT primer (5′- /5Phos/CAGAGCNNNNNNNN[10bp barcode]TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3′, where “N” is any base; IDT) and 2 µL 10 mM dNTP mix (Thermo), denatured at 55°C for 5 min and immediately placed on ice. 14 µL of first-strand reaction mix, containing 8 µL 5X Superscript IV First-Strand Buffer (Invitrogen), 2 µl 100 mM DTT (Invitrogen), 2 µl SuperScript IV reverse transcriptase (200 U/μl, Invitrogen), 2 μL RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen), was then added to each well. Reverse transcription was carried out by incubating plates by gradient temperature (4°C 2 minutes, 10°C 2 minutes, 20°C 2 minutes, 30°C 2 minutes, 40°C 2 minutes, 50°C 2 minutes and 55°C 10 minutes). After ligation reaction, 60µL nuclei dilution buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 1% BSA) was added into each well. Nuclei from all wells were pooled together and spun down at 500xg for 10min. Nuclei were then resuspended in nuclei wash buffer and redistributed into another four 96-well plates with each well including 4µL T4 ligation buffer (NEB), 2µL T4 DNA ligase (NEB), 4µL Betaine solution (5M, Sigma-Aldrich), 6µL nuclei in nuclei wash buffer, 8µL barcoded ligation adaptor (100uM, 5’- GCTCTG[9bp or 10bp barcode A]/ideoxyU/ACGACGCTCTTCCGATCT[reverse complement of barcode A]-3’) and 16µL 40% PEG 8000 (Sigma-Aldrich). The ligation reaction was done at 16°C for 3 hours. After RT reaction, 60µL nuclei dilution buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 1% BSA) was added into each well. Nuclei from all wells were pooled together and spun down at 600xg for 10min. Nuclei were washed once with nuclei wash buffer and filtered with 1ml Flowmi cell strainer (Flowmi) twice, counted and redistributed into eight 96-well plates with each well including 2,500 nuclei in 5µL nuclei wash buffer and 5µL elution buffer (Qiagen). 1.33 μl mRNA Second Strand Synthesis buffer (NEB) and 0.66 μl mRNA Second Strand Synthesis enzyme (NEB) were then added to each well, and second strand synthesis was carried out at 16°C for 180 min. For tagmentation, each well was mixed with 11 μL Nextera TD buffer (Illumina) and 1 μL i7 only TDE1 enyzme (62.5nM, Illumina), and then incubated at 55°C for 5 min to carry out tagmentation. The reaction was then stopped by adding 24 μL DNA binding buffer (Zymo) per well and incubating at room temperature for 5 min. Each well was then purified using 1.5x AMPure XP beads (Beckman Coulter). In the elution step, each well was added with 8µL nuclease free water, 1µL 10X USER buffer (NEB), 1µL USER enzyme (NEB) and incubated at 37°C for 15 min. Another 6.5µL elution buffer was added into each well. The AMPure XP beads were removed by magnetic stand and the elution product was transferred into a new 96-well plate. For PCR amplification, each well (16µL product) was mixed with 2μL of 10 μM indexed P5 primer (5′-AATGATACGGCGACCACCGAGATCTACAC[i5]ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′; IDT), 2 μL of 10 μM P7 primer (5′-CAAGCAGAAGACGGCATACGAGAT[i7]GTCTCGTGGGCTCGG-3′, IDT), and 20 μL NEBNext High-Fidelity 2X PCR Master Mix (NEB). Amplification was carried out using the following program: 72°C for 5 min, 98°C for 30 sec, 12-14 cycles of (98°C for 10 sec, 66°C for 30 sec, 72°C for 1 min) and a final 72°C for 5 min.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

sci-RNA-seq3 library

Data processing

For sc-RNA-seq processing, read alignment and gene count matrix generation for single cell RNA-seq of co-assay was the same with sci-RNA-seq ("J. Cao et al., Comprehensive single cell transcriptional profiling of a multicellular organism by combinatorial indexing. bioRxiv, 104844 (2017).") with minor modifications: Reads were mapped to a chimeric reference genome of human (hg19) and mouse (mm10) for RNA-seq from A594, HEK293T and NIH/3T3 cells, and mouse (mm10) only for RNA-seq from mouse kidney experiment with STAR/v 2.5.2b, with gene annotations (GENCODE V19 for human; GENCODE VM11 for mouse). For mixed-species experiment, the percentage of uniquely mapping reads for genomes of each species was calculated. Cells with over 80% of UMIs assigned to one species were regarded as species-specific cells, with the remaining cells classified as mixed cells or “collisions”.
Genome_build: mouse reference genome (mm10)
Supplementary_files_format_and_content: Processed data files include a cell annotation csv file, gene annotation csv file, and a gene count sparse matrix file
Supplementary_files_format_and_content: Cell annotation file: sample is cell id of each single cell with the reverse transcription and ligation barcode attached; tsne_1 and tsne_2 are cell coordinates after t-SNE dimension reduction.
Supplementary_files_format_and_content: Gene annotation file including gene id, gene type and gene short name.
Supplementary_files_format_and_content: A sparse matrix file: each row corresponds to gene id (with gene information in the gene_annotation.csv; each column corresponds to each cell (with cell source information in the cell_annotation.csv); each value in the matrix corresponds to UMI count.

Submission date

Sep 13, 2018

Last update date

Feb 20, 2019

Contact name

Junyue Cao

E-mail(s)

[email protected]

Organization name

University of Washington

Department

Department of Genome Sciences