title: 203219760089_R08C01

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title: 203219760185_R01C01

status: Public on Oct 14 2024

type: genomic

city: Detroit

last: Pilsner

state: MI

address: 275 E Hancock St

department: Department of Obstetrics and Gynecology

country: USA

institute: Wayne State University

description:
sperm DNA methylation
Processed_methylation_data_matrix_LIFE.RData

hyb_protocol: bisulphite converted DNA was amplified, fragmented and hybridised to  Illumina Infinium HumanMethylationEPIC BeadChip using standard Illumina protocol

scan_protocol: Arrays were imaged using BeadArray Reader using standard recommended Illumina scanner setting

data_processing:
Sperm DNA methylation was assessed using Illumina's EPIC Infinium Methylation Beadchip. Raw idat files were imported into R and within-array normalization and dye-bias equalization of Type I and Type II probes was performed using normal-exponential convolution method (Noob) via  minfi package. Batch effects were corrected with the ComBat function in the sva package while cross-hybridizing probes were removed using the DMRcate package. probes with detection pvalue >0.01 were also exluced prior to downstream analysis.
Processed matrix contains the NOOB-normalized beta values representing the percent CpG methylation for all the CpGs that passed the preprocessing method.

molecule: genomic DNA

organism: Homo sapiens

source_name: normal sperm sample

label_protocol: Standard Illumina Protocol

extract_protocol: Standard Illumina Protocol

treatment_protocol: Sperm from semen was extracted using gradient method and purified DNA was isolated our previously developed method of beads, RLT and TCEPS

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value: LIFE

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title: 203219760185_R03C01

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description:
sperm DNA methylation
Processed_methylation_data_matrix_EARTH.RData

value: EARTH

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tag: minp

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tag: mep


title: 205049770086_R06C01

tag: specific gravity

tag: batch

tag: mehp

tag: mecpp

tag: mehhp

tag: meohp

tag: mcpp

tag: mhbp

tag: mhibp

tag: mhinch

tag: mcoch


title: 205049770045_R03C01

tag: tissue

tag: cohort

tag: bmi

tag: smoking status

tag: race

tag: creatinine

tag: mcmhp

tag: mop

tag: mbp

tag: mibp

tag: mcnp

tag: mcop


title: 205049770045_R02C01

tag: specific gravity

tag: batch

tag: mehp

tag: mecpp

tag: mehhp

tag: meohp

tag: mcpp

tag: mchp

tag: mbzp

tag: minp

tag: mmp

tag: mep


title: 205049770025_R08C01

tag: tissue

tag: cohort

tag: bmi

tag: race

tag: creatinine

tag: mcmhp

tag: mop

tag: mhbp

tag: mhibp

tag: mhinch

tag: mcoch


title: 205049770045_R01C01

tag: specific gravity

tag: batch

tag: mehp

tag: mecpp

tag: mehhp

tag: meohp

tag: mcpp

tag: mbp

tag: mibp

tag: mcnp

tag: mcop


title: 205049770025_R07C01

tag: tissue

tag: cohort

tag: bmi

tag: race

tag: creatinine

tag: mcmhp

tag: mchp

tag: mbzp

tag: minp

tag: mmp

tag: mep


title: 205049770025_R04C01

tag: specific gravity

tag: batch

tag: mehp

tag: mecpp

tag: mehhp

tag: meohp

tag: mop

tag: mhbp

tag: mhibp

tag: mhinch

tag: mcoch


title: 205049770025_R05C01

tag: tissue

tag: cohort

tag: bmi

tag: race

tag: creatinine

tag: mcmhp

tag: mcpp

tag: mbp

tag: mibp

tag: mcnp

tag: mcop


title: 205049770082_R02C01

tag: specific gravity

tag: batch

tag: mehp

tag: mecpp

tag: mehhp

tag: meohp

tag: mchp

tag: mbzp

tag: minp

tag: mmp

tag: mep


title: 205049770025_R01C01

tag: tissue

tag: cohort

tag: bmi

tag: creatinine

tag: mcmhp

tag: mop

tag: mhbp

tag: mhibp

tag: mhinch

tag: mcoch


title: 205049770017_R08C01

tag: race

tag: specific gravity

tag: batch

tag: mehp

tag: mecpp

tag: mehhp

tag: mcpp

tag: mbp

tag: mibp

tag: mcnp

tag: mcop


title: 205049770025_R03C01

tag: tissue

tag: cohort

tag: bmi

tag: creatinine

tag: mcmhp

tag: meohp

tag: mchp

tag: mbzp

tag: minp

tag: mmp

tag: mep


title: 205049770025_R02C01

tag: race

tag: specific gravity

tag: batch

tag: mehp

tag: mecpp

tag: mehhp

tag: mop

tag: mhbp

tag: mhibp

tag: mhinch

tag: mcoch


title: 205049770017_R07C01

tag: tissue

tag: cohort

tag: bmi

tag: creatinine

tag: mcmhp

tag: meohp

tag: mcpp

tag: mbp

tag: mibp

tag: mcnp

tag: mcop


title: 205049770017_R06C01

tag: race

tag: specific gravity

tag: batch

tag: mehp

tag: mecpp

tag: mehhp

tag: mchp

tag: mbzp

tag: minp

tag: mmp

tag: mep


title: 205049770017_R05C01

tag: tissue

tag: cohort

tag: bmi

tag: creatinine

tag: mcmhp

tag: meohp

tag: mop

tag: mhbp

tag: mhibp

tag: mhinch

tag: mcoch


title: 205049770017_R03C01

tag: specific gravity

tag: batch

tag: mehp

tag: mecpp

tag: mehhp

tag: mcpp

tag: mbp

tag: mibp

tag: mcnp

tag: mcop


title: 205049770017_R02C01

tag: tissue

tag: cohort

tag: bmi

tag: race

tag: creatinine

tag: mcmhp

tag: mchp

tag: mbzp

tag: minp

tag: mmp

tag: mep


title: 205030240117_R05C01

tag: specific gravity

tag: batch

tag: mehp

tag: mecpp

tag: mehhp

tag: meohp

tag: mop

tag: mhbp

tag: mhibp

tag: mhinch

tag: mcoch


title: 205030240117_R03C01

tag: tissue

tag: cohort

tag: bmi

tag: race

tag: creatinine

tag: mcmhp

tag: mcpp

tag: mbp

tag: mibp

tag: mcnp

tag: mcop


title: 205049770017_R01C01

tag: specific gravity

tag: batch

tag: mehp

tag: mecpp

tag: mehhp

tag: meohp

tag: mchp

tag: mbzp

tag: minp

tag: mmp

tag: mep


title: 205030240117_R04C01

tag: tissue

tag: cohort

tag: bmi

tag: race

tag: creatinine

tag: mcmhp

tag: mop

tag: mhbp

tag: mhibp

tag: mhinch

tag: mcoch


title: 205030240117_R02C01

tag: specific gravity

tag: batch

tag: mehp

tag: mecpp

tag: mehhp

tag: meohp

tag: mcpp

tag: mbp

tag: mibp

tag: mcnp

tag: mcop


title: 205030240006_R06C01

tag: tissue

tag: cohort

tag: bmi

tag: race

tag: creatinine

tag: mcmhp

tag: mchp

tag: mbzp

tag: minp

tag: mmp

tag: mep


title: 205030240117_R01C01

tag: specific gravity

tag: batch

tag: mehp

tag: mecpp

tag: mehhp

tag: meohp

tag: mop

tag: mhbp

tag: mhibp

tag: mhinch

tag: mcoch


title: 205030240006_R07C01

tag: tissue

tag: cohort

tag: bmi

tag: race

tag: creatinine

tag: mcmhp

tag: mcpp

tag: mbp

tag: mibp

tag: mcnp

tag: mcop


title: 205030240006_R05C01

tag: specific gravity

tag: batch

tag: mehp

tag: mecpp

tag: mehhp

tag: meohp

tag: mchp

tag: mbzp

tag: minp

tag: mmp

tag: mep


title: 205049770025_R06C01

tag: tissue

tag: cohort

tag: bmi

tag: race

tag: creatinine

tag: mcmhp

tag: mop

tag: mhbp

tag: mhibp

tag: mhinch

tag: mcoch


title: 205030240006_R02C01

tag: specific gravity

tag: batch

tag: mehp

tag: mecpp

tag: mehhp

tag: meohp

tag: mcpp

tag: mbp

tag: mibp

tag: mcnp

tag: mcop


title: 205030230141_R07C01

tag: tissue

tag: cohort

tag: bmi

tag: race

tag: creatinine

tag: mcmhp

tag: mchp

tag: mbzp

tag: minp

tag: mmp

tag: mep


title: 205030230141_R06C01

tag: specific gravity

tag: batch

tag: mehp

tag: mecpp

tag: mehhp

tag: meohp

tag: mop

tag: mhbp

tag: mhibp

tag: mhinch

tag: mcoch


title: 205030230141_R05C01

tag: tissue

tag: cohort

tag: bmi

tag: race

tag: creatinine

tag: mcmhp

tag: mcpp

tag: mbp

tag: mibp

tag: mcnp

tag: mcop


title: 205030230141_R02C01

tag: specific gravity

tag: batch

tag: mehp

tag: mecpp

tag: mehhp

tag: meohp

tag: mchp

tag: mbzp

tag: minp

tag: mmp

tag: mep


title: 205030230141_R04C01

tag: tissue

tag: cohort

tag: bmi

tag: race

tag: creatinine

tag: mcmhp

tag: mop

tag: mhbp

tag: mhibp

tag: mhinch

tag: mcoch


title: 205030230141_R03C01

tag: specific gravity

tag: batch

tag: mehp

tag: mecpp

tag: mehhp

tag: meohp

tag: mcpp

tag: mbp

tag: mibp

tag: mcnp

tag: mcop


title: 205030230141_R01C01

tag: tissue

tag: cohort

tag: bmi

tag: race

tag: creatinine

tag: mcmhp

tag: mchp

tag: mbzp

tag: minp

tag: mmp

tag: mep


title: 205030230139_R07C01

tag: specific gravity

tag: batch

tag: mehp

tag: mecpp

tag: mehhp

tag: meohp

tag: mop

tag: mhbp

tag: mhibp

tag: mhinch

tag: mcoch


title: 205030230139_R06C01

tag: tissue

tag: cohort

tag: bmi

tag: race

tag: creatinine

tag: mcmhp

tag: mcpp

tag: mbp

tag: mibp

tag: mcnp

tag: mcop


title: 205030240117_R06C01

tag: specific gravity

tag: batch

tag: mehp

tag: mecpp

tag: mehhp

tag: meohp

tag: mchp

tag: mbzp

tag: minp

tag: mmp

tag: mep


title: 205030230139_R05C01

tag: tissue

tag: cohort

tag: bmi

tag: race

tag: creatinine

tag: mcmhp

tag: mop

tag: mhbp

tag: mhibp

tag: mhinch

tag: mcoch


title: 205030240006_R08C01

tag: specific gravity

tag: batch

tag: mehp

tag: mecpp

tag: mehhp

tag: meohp

tag: mcpp

tag: mbp

tag: mibp

tag: mcnp

tag: mcop


title: SMC Donor6, Quiescent, IgG

type: SRA

city: Charlottesville

first: Nicolas

last: Barbera

state: Virginia

address: 1335 Lee Street

department: Department of Genome Sciences

institute: University of Virginia

library_source: genomic

data_processing:
Basecalls were performed using bcl2fastq v2.17
Trim Galore was utilized to remove contaminant adapters and for read-quality trimming
Paired-end reads were aligned to the human reference genome (HG38) using Bowtie2 (v2.2.6) ( --local --very-sensitive --no-unal --no-mixed --no-discordant --phred33 -I 10 -X 700)
Picard MarkDuplicates tool was utilized to mark and remove PCR duplicates in each sample, and reads with less than 20 MAPQ score were filtered
Filtered bam files were converted to bed files with bedtools bamtobed and regions longer than 1000 bp were removed. ENCODE blacklisted regions on hg38 were removed.
Bedtools genomecov was used to convert bed files into bedgraph files
Assembly: GRCh37
Supplementary files format and content: bedgraph
Library strategy: CUT&RUN

source_name: Ascending Aorta

growth_protocol: HCASMCs were cultured in either Lonza Complete SmBM media or FBS Free SmBM media on gelatin-coated dishes

extract_protocol:
Cleavage Under Targets and Release Using Nuclease (CUT&RUN) sequencing was performed using the assay kit from Cell Signaling Technology (cat# 86652). Briefly, SMCs were cultured in both quiescent and proliferative conditions, to an average of 1 million cells per plate. Cells were harvested with trypsin and washed with a wash buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, and protease inhibitor cocktail). Cells were permeabilized with digitonin and incubated with activated concanavalin A magnetic beads. Cell-bead conjugates were then resuspended in 100 µL of digitonin buffer (wash buffer with 2.5% digitonin solution) containing 2 uL of Anti-Tri-Methyl-Histone H3 (Lys4) (CST, #9733), 1 uL of Anti-Acetyl-Histone H3 (Lsy 27) (CST, #8173), 2 uL of Anti-H2BK20ac (#34156), or rabbit IgG (CST, #66362), rotated overnight at 4°C, resuspended in 250 µL of antibody buffer and 7.5 µL of the pAG-MNase enzyme (#57813, CST), followed by the rotation at 4°C for 1 h. After MNase-induced cleavage, genomic DNA fragments were extracted using DNA spin columns (CST, #14209).
Samples were prepared for sequencing with the DNA Library Prep Kit for Illumina (CST, #56795) combined with Multiplex Oligos for Illumina® (Dual Index Primers) (CST, #47538). The adaptor was diluted 1:125 to avoid contamination. The PCR enrichment step ran 20 cycles to amplify the adaptor-ligated CUT&RUN DNA.

tag: tissue

value: Ascending Aorta

tag: cell type

value: Smooth Muscle Cell

tag: Sex

value: female

tag: treatment

value: Quiescent


title: SMC Donor6, Quiescent, H2BK20ac


title: 205030240006_R03C01

tag: cohort

tag: bmi

tag: race

tag: creatinine

tag: mcmhp

tag: mop

tag: mchp

tag: mbzp

tag: minp

tag: mmp

tag: mep

tag: mhbp

tag: mhibp

tag: mhinch


title: 205030240006_R04C01

tag: tissue

tag: specific gravity

tag: batch

tag: mehp

tag: mecpp

tag: mehhp

tag: meohp

tag: mcpp

tag: mbp

tag: mibp

tag: mcnp

tag: mcop

tag: mcoch


title: SMC Donor1, Quiescent, IgG

tag: cell type

tag: Sex

value: male

tag: treatment


title: SMC Donor2, Quiescent, IgG


title: SMC Donor5, Quiescent, H2BK20ac


title: SMC Donor4, Quiescent, H2BK20ac


title: SMC Donor3, Quiescent, H2BK20ac


title: SMC Donor5, Quiescent, IgG


title: SMC Donor2, Quiescent, H2BK20ac


title: SMC Donor4, Quiescent, IgG


title: 205030240117_R08C01

tag: cohort

tag: bmi

tag: race

tag: creatinine

tag: mcmhp

tag: mop

tag: mchp

tag: mbzp

tag: minp

tag: mmp

tag: mep

tag: mhbp

tag: mhibp

tag: mhinch


title: SMC Donor1, Quiescent, H2BK20ac

tag: tissue

tag: cell type


title: 205030230139_R08C01

tag: specific gravity

tag: batch

tag: mehp

tag: mecpp

tag: mehhp

tag: meohp

tag: mcpp

tag: mbp

tag: mibp

tag: mcnp

tag: mcop

tag: mcoch


title: SMC Donor3, Quiescent, H3K27ac

tag: Sex

tag: treatment


title: SMC Donor2, Quiescent, H3K27ac


title: SMC Donor1, Quiescent, H3K27ac


title: SMC Donor6, Quiescent, H3K27ac


title: SMC Donor5, Quiescent, H3K4me3


title: SMC Donor4, Quiescent, H3K4me3


title: SMC Donor6, Quiescent, H3K4me3


title: SMC Donor4, Quiescent, H3K27ac


title: SMC Donor3, Quiescent, H3K4me3


title: SMC Donor5, Quiescent, H3K27ac


title: SMC Donor3, Quiescent, IgG


title: SMC Donor2, Quiescent, H3K4me3


title: SMC Donor6, Proliferative, IgG

value: Proliferative


title: SMC Donor1, Quiescent, H3K4me3


title: SMC Donor5, Proliferative, IgG


title: SMC Donor4, Proliferative, IgG


title: SMC Donor3, Proliferative, H2BK20ac


title: SMC Donor2, Proliferative, H2BK20ac


title: SMC Donor5, Proliferative, H2BK20ac


title: SMC Donor1, Proliferative, IgG


title: SMC Donor6, Proliferative, H2BK20ac


title: SMC Donor3, Proliferative, IgG


title: SMC Donor4, Proliferative, H2BK20ac


title: SMC Donor3, Proliferative, H3K27ac


title: SMC Donor1, Proliferative, H3K27ac


title: SMC Donor5, Proliferative, H3K4me3


title: SMC Donor3, Proliferative, H3K4me3


title: SMC Donor2, Proliferative, H3K27ac


title: SMC Donor2, Proliferative, H3K4me3


title: SMC Donor1, Proliferative, H3K4me3


title: HT29 Colonospheres Control 3

city: NEW ORLEANS

first: Suzana

middle: D

last: Savkovic

state: LA

address: 1430 Tulane Ave JBJ 315

institute: Tulane University

description: HT29 colonospheres DGAT1&2 inhibitors vs Control. Summary. Genes.xlsx

library_source: transcriptomic

data_processing:
Paired-end reads were aligned to hg38 genome using STAR(v.2.6.1d).
Abundance levels were calculated using Kallisto(v.0.46.0) a
Differential expression testing performed using DESeq2.
Assembly: mouse, mm10 and human, hg38
Supplementary files format and content: Excel files with TPMs and results of differential expression testing from DESeq2

molecule: total RNA

source_name: cell line

extract_protocol:
Zymogen, Dnase Treated
High Output Paired End Read

value: cell line

tag: cell line

value: Colonospheres

tag: genome build

value: Colonospheres were treated with serum-free media


title: HT29 Colonospheres DGAT1/2 Inhs. 1

tag: tissue

tag: cell type

value: Colonospheres were treated with DGAT1/2 inhibitors.


title: HT29 Colonospheres DGAT1/2 Inhs. 3


title: HT29 DGAT1/2 Inhs. 5

description: HT29 cells DGAT1&2 Inhibitors Vs Control. Summary. Genes.xlsx

value: Colon Cancer Cells

value: Cells were treated with DGAT1/2 inhibitors.


title: HT29 Colonospheres Control 2


title: HT29 Colonospheres DGAT1/2 Inhs. 2


title: HT29 Colonospheres Control 1


title: SMC Donor2, Proliferative, IgG


title: HT29 DGAT1/2 Inhs. 4


title: SMC Donor1, Proliferative, H2BK20ac


title: SMC Donor6, Proliferative, H3K27ac


title: HT29 DGAT1/2 Inhs. 1


title: HT29 DGAT1/2 Inhs. 3


title: HT29 Control 3

value: Cells were treated with serum-free media


title: HT29 DGAT1/2 Inhs. 2


title: SMC Donor4, Proliferative, H3K27ac


title: HT29 Control 2


title: SMC Donor6, Proliferative, H3K4me3


title: SFSWAP knockdown in UPF1 knockdown background - Biological Replicate 2  (TG-treated)

city: Dallas

first: Nicholas

middle: K

last: Conrad

state: Texas

address: 6000 Harry Hines Blvd

department: Microbiology

institute: UT Southwestern Medical Center

data_processing:
fastq files were trimmed with Cutadapt v 1.9.1
Trimmed reads were aligned to a custom reference genome based on RefSeq GCF_000001405.34/GRCh38.p8 (with additional entries corresponding to decoy exons; see paper) using STAR v 2.7 in twoPassBasic mode
Alternate splicing analysis was performed using rMATS v 4.3.0
Assembly: RefSeq GCF_000001405.34/GRCh38.p8 with additional custom entries corresponding to decoy exons (see paper)
Supplementary files format and content: TG.SE.MATS.JCEC.txt - text file; contains skipped exon alternate splicing events output from rMATS (JCEC model) for non-siUPF1-treated samples (Sample 1 corresponds to siNT and Sample 2 corresponds to siSFSWAP)
Supplementary files format and content: TG.RI.MATS.JCEC.txt - text file; contains skipped exon alternate splicing events output from rMATS (JCEC model) for non-siUPF1-treated samples (Sample 1 corresponds to siNT and Sample 2 corresponds to siSFSWAP)
Supplementary files format and content: UPF1.TG.SE.MATS.JCEC.txt - text file; contains skipped exon alternate splicing events output from rMATS (JCEC model) for siUPF1-treated samples (Sample 1 corresponds to siNT/siUPF1 and Sample 2 corresponds to siSFSWAP/siUPF1)
Supplementary files format and content: UPF1.TG.RI.MATS.JCEC.txt - text file; contains skipped exon alternate splicing events output from rMATS (JCEC model) for siUPF1-treated samples (Sample 1 corresponds to siNT/siUPF1 and Sample 2 corresponds to siSFSWAP/siUPF1)

molecule: polyA RNA

growth_protocol: HEK293A-TOA cells were grown in DMEM supplemented with Tet-free FBS, glutamate and penicillin-streptomycin.

extract_protocol:
Total RNA was isolated using Zymo Direct-zol RNA Miniprep Kits and 1 μg of RNA was used for polyA RNA isolation.
Sequencing libraries were prepared with KAPA mRNA HyperPrep Kit using index primers from NEB (NEBNext Multiplex Oligos for Illumina - Index Primers Set 2).

treatment_protocol: Cells were transfected with 40 nM siRNA (mixture of 2 different siRNAs, see paper for details) against SFSWAP and 30 nM siRNA against UPF1 (if applicable), split on the next day and grown for 4 additional days. Cells were treated with 1 μM TG for 6 hours before harvesting in trizol.

value: 1 μM TG for 6 hours


title: SFSWAP knockdown - Biological Replicate 1  (TG-treated)


title: SFSWAP knockdown in UPF1 knockdown background - Biological Replicate 1  (TG-treated)


title: SFSWAP knockdown - Biological Replicate 3

data_processing:
fastq files were trimmed with Cutadapt v 1.9.1
Trimmed reads were aligned to GENCODE GRCh38 v40 genome using STAR v 2.7 in twoPassBasic mode
Alternate splicing analysis was performed using rMATS v 4.3.0
Assembly: GENCODE GRCh38 v40
Supplementary files format and content: SE.MATS.JC.txt - text file; contains skipped exon alternate splicing events output from rMATS (JC model) (Sample 1 corresponds to siNT and Sample 2 corresponds to siSFSWAP)
Supplementary files format and content: RI.MATS.JC.txt - text file; contains retained intron alternate splicing events output from rMATS (JC model) (Sample 1 corresponds to siNT and Sample 2 corresponds to siSFSWAP)

extract_protocol:
Total RNA was isolated using Zymo Direct-zol RNA Miniprep Kits and 1 μg of RNA was used for polyA RNA isolation.
Sequencing libraries were prepared with KAPA mRNA HyperPrep Kit using index primers from NEB (NEBNext Multiplex Oligos for Illumina - Index Primers Set 1).

treatment_protocol: Cells were transfected with 40 nM siRNA (mixture of 2 different siRNAs, see paper for details) against SFSWAP, split on the next day and grown for 4 additional days.


title: Non-target - Biological Replicate 2  (TG-treated)


title: Non-target in UPF1 knockdown background - Biological Replicate 1 (TG-treated)


title: SFSWAP knockdown - Biological Replicate 2


title: SFSWAP knockdown in UPF1 knockdown background - Biological Replicate 3  (TG-treated)


title: Non-target - Biological Replicate 3  (TG-treated)


title: Non-target - Biological Replicate 1 (TG-treated)


title: SFSWAP knockdown - Biological Replicate 3  (TG-treated)


title: Non-target in UPF1 knockdown background - Biological Replicate 2  (TG-treated)


title: SFSWAP knockdown - Biological Replicate 1


title: Non-target - Biological Replicate 2


title: Non-target - Biological Replicate 1


title: HT29 Control 1


title: Non-target in UPF1 knockdown background - Biological Replicate 3  (TG-treated)


title: D5-Teff-Stim-AAVS1-KO-Input_ChIP

city: San Francisco

first: Alexander

last: Marson

state: CA

address: 1650 Owens St

institute: UCSF Gladstone Institutes

data_processing:
Reads were trimmed to remove adapters and low-quality sequences and aligned to the hg38 and mm10 reference genome assemblies with bwa65 (v0.7.17-r1188) before filtering to remove duplicates and low-quality alignments including to problematic genomic regions66 using the nf-core/chipseq pipeline67 (v2.0.0, doi: 10.5281/zenodo.3240506) with default parameters.
Normalization to mouse spike-in chromatin was performed by scaling counts to the quotient of the ratios of human:mouse ChIP reads and human:mouse input reads as described in Orlando, D. A. et al. Cell Rep 9, 1163–1170 (2014).
MED12 peaks were identified using bam files from AAVS1 KO samples using the MED12 KO samples as the background control for MACS2 callpeak -q 0.05
Assembly: GRCh38
Supplementary files format and content: MACS2 output narrow peak files are provided for non-control samples containing called peaks. For MED12 peaks, the AAVS1 KO sample of the matching condition was used to call the peak instead of the input sample
Supplementary files format and content: bigwigs are provided for all samples and are normalized to mouse chromatin spikein and input unless specified as not normalized (input samples)

source_name: Blood

growth_protocol: CD4+ effector T cells were isolated from fresh Peripheral Blood Leukopaks (STEMCELL Technologies, #70500) from healthy human donors, after institutional review board–approved informed written consent (STEMCELL Technologies). The contents of the Leukopaks were washed twice with a 1X volume of EasySep buffer (DPBS, 2% fetal Bovine Serum (FBS), 1mM pH 8.0 EDTA) using centrifugation. The washed cells were resuspended at 200E6 cells/mL in EasySep buffer and isolated with the EasySep™ Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit (STEMCELL Technologies, #18063), according to the manufacturer’s protocol. Teffs were seeded at 1x106 cells/mL in RPMI-1640 supplemented with 10% FCS, 2 mM L-Glutamine (Fisher Scientific #25030081), 10 mM HEPES (Sigma, #H0887-100ML), 1X MEM Non-essential Amino Acids (Fisher, #11140050), 1 mM Sodium Pyruvate (Fisher Scientific #11360070), 100 U/mL Penicillin-Streptomycin (Sigma, #P4333-100ML), and 50 U/mL IL-2 (Amerisource Bergen, #10101641). Cells were then stimulated with ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator (STEMCELL Technologies, #10990) at 6.25 uL/mL for Teff. Following activation and electroporation, cells were split 1:2 every 48 hours to maintain an approximate density of 1x106 cells/mL and supplemented with respective doses of IL-2.

extract_protocol:
A portion of edited Teffs were re-stimulated with ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator (STEMCELL Technologies, #10990) 10 days following isolation and collected 48 hours later. Up to 1-2 x 106 Teffs were cross-linked in PBS with 1% methanol-free formaldehyde (Thermo 28908) for 10 min at 18-22ºC followed by quenching in glycine at 125 mM final concentration. Cross-linked cell pellets were snap-frozen in liquid nitrogen and stored at –80ºC. Nuclei were isolated from thawed, cross-linked cells via sequential lysis in LB1 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% IGEPAL CA-360, and 0.25% Triton X-100), LB2 (10 mM Tris-HCl pH 8, 200 mM NaCl, 1 mM EDTA, and 0.5 mM EGTA), and LB3 (10 mM Tris-HCl pH 8, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% sodium deoxycholate [NaDOC], and 0.5% N-laurylsarcosine) supplemented with 0.5 mM phenylmethylsulfonyl fluoride (PMSF, Sigma P7626) and 0.5X protease inhibitor cocktail (PIC, Sigma P8340). Chromatin was sheared on a Covaris E220 focused ultrasonicator using 1 mL milliTubes (Covaris 520128) with 140W peak incident power, 5% duty factor, 200 cycles per burst, 6ºC temperature setpoint (minimum 3ºC, maximum 9ºC), fill level 10, and time 12-14 min to obtain target size 200-700 bp. Formaldehyde cross-linked, sheared mouse CD8 T cell chromatin was spiked in at 2.5% of human Teff chromatin based on fluorometric (Qubit, Thermo Q33238) or OD260 (Nanodrop, Thermo 912A1099) quantification. Triton X-100 was added to final concentration of 1% prior to immunoprecipitation for 16 h at 4ºC with 2-8 µg of indicated antibodies (Supplemental table 21) bound to a 1:1 mixture of protein A and protein G magnetic beads (Thermo 10001D and 10003D). Bead-bound antibody-chromatin complexes were sequentially washed three times with Wash Buffer 1 (20 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium dodecyl sulfate [SDS], and 0.1% NaDOC), twice with Wash Buffer 2 (20 mM Tris-HCl pH 8, 500 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, and 0.1% NaDOC), twice with Wash Buffer 3 (20 mM Tris-HCl pH 8, 250 mM LiCl, 1 mM EDTA, 0.5% IGEPAL CA-360, and 0.5% NaDOC), twice with TET (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.2% Tween-20), and once with TE0.1 (10 mM Tris-HCl pH 8, 0.1 mM EDTA, 0.5 mM PMSF, and 0.5X PIC) supplemented with 0.5 mM PMSF and 0.5X PIC.
Beads were resuspended in TT (10 mM Tris-HCl pH 8, 0.05% Tween-20) prior to on-bead library preparation using the NEBNext Ultra II DNA Library Prep Kit (NEB E7370L) as described previously64. ChIP-seq libraries were multiplexed for paired-end (2 x 50bp) sequencing on an Illumina NextSeq 2000 instrument.

treatment_protocol: Guide loaded Cas9 RNPs were assembled with custom crRNAs (Dharmacon) which were resuspended in IDT duplex buffer (IDT, #11-01-03-01) at 160uM. Dharmacon Edit-R CRISPR-Cas9 Synthetic tracrRNA (Dharmacon, #U-002005-20) also resuspended in Nuclease Free Duplex Buffer at 160uM was combined at a 1:1 molar ratio in a 96 well plate and incubated at 37°C for 30 minutes. Single-stranded donor oligonucleotides (ssODN; sequence: TTAGCTCTGTTTACGTCCCAGCGGGCATGAGAGTAACAAGAGGGTGTGGTAATATTACGGTACCGAGCACTATCGATACAATATGTGTCATACGGACACG, 100uM stock) was added to the complex at a 1:1 molar ratio and incubated at 37°C for 5 minutes. Finally, Cas9 protein (MacroLab, Berkeley, 40 µM stock) was added at a 1:2 molar ratio and incubated at 37°C for 15 minutes. The resulting RNPs were frozen at -80°C until the day of electroporation and were thawed to room temperature prior to use. 48 hours following T cell activation, the cells were pelleted at 100x g for 10 minutes and resuspended in room temperature P3 Primary Cell Nucleofector Buffer (Lonza, catalog no. V4XP-3032) at 1.5E6 cells per 17.8 µL. 1.5E6 cells were transferred to each RNP containing well and mixed gently. 25 µL of the combined RNP cell solution was transferred to a 96-well electroporation cuvette plate (Lonza, #VVPA-1002) and nucleofected with pulse code DS-137. Immediately following electroporation, the cells were gently resuspended in 90 µL warmed media and incubated at 37 C for 15 minutes. After recovery, the cells were cultured in flasks at 1x106 cells/mL for the duration of the experiment.

value: Blood

tag: chip antibody

value: Input

value: CD4+ Teff

tag: genotype

tag: condition

value: Stim

tag: donor


title: D4-Teff-Stim-AAVS1-KO-Input_ChIP


title: AKT2, 0 mM, SSII

city: New York

first: Samie

last: Jaffrey

state: New York

address: 1300 York Ave

department: Pharmacology

institute: Weill Cornell Medicine

description:
Duration: 0;temperature: 37C;buffer: SH-SY5Y, 200 mM Bicine pH 8.3, ;ligand: N/A
AKT2 untreated

data_processing:
Sequencing data was processed using ShapeMapperV2.2 (https://github.com/Weeks-UNC/shapemapper2)
For HeLa 18s rRNA experiments, sequencing data was aligned to 12 unique 18S amplicons defined by the unique combinations of 4-nucleotide forward and 7-nucleotide reverse barcode sequences inserted during step 1 PCR. Sequencing data alignment was performed with ShapeMapperV2.2 with the following settings: --amplicon --output-parsed --output-aligned-reads --nproc 15 --output-counted-mutations --min-mutation-separation 0.
For Spinach, pUG, and AKT2 experiments, sequencing data was aligned to a single fasta sequence with ShapeMapperV2.2 with the following settings: --amplicon --dms --output-parsed - -min-mutation-separation 2. Deletions are ambiguously aligned and therefore ignored by invoking the --dms option.
Misincorporation signatures were calculated by aligning the data with ShapeMapperV2.2 with the following settings: --amplicon --output-counted-mutations --min-mutation-separation 0.
Counts of misincorporation types for each position within an RNA are tabulated by the --output-counted-mutations field. A minimum misincorporation separation distance of 0 was chosen to capture the full range of misincorporation types generated in BASH MaP and DMS MaP experiments.
ShapeMapper alignment with --output-parsed produces a .mut file which encodes the misincorporations within a sequencing read as a bitvector where 0 represents no misincorporation and 1 represents a misincorporation. This .mut file was then input into the program RingMapper which identifies positions with statistically high rates of co-occurring misincorporations7. The output of RingMapper was processed with the custom python script ring_pair_to_heatmap.py which filters all negative correlations and creates a square matrix which was then visualized in Prism GraphPad.
For M2 Spinach and pUG experiments, sequencing data was aligned to a single fasta sequence with ShapeMapperV2.2 with the following settings: --amplicon --dms --output-parsed --min- mutation-separation 2. The output parsed mutation file was then processed with the custom script mut_to_simple.py which retains only sequencing reads that cover the entire length of the RNA. Then the output .simple file was used to calculate a unique mutational profile for each point mutant with the script simple_to_M2_map.py. This script produces a square matrix with length equal to the length of the RNA and displays how often two mutations co-occur on the same sequencing read. To better visualize changes in nucleotide reactivity given a mutation at a certain position, Z-score normalization was performed with the script M2_Map_to_Zscores.py.
The normalized matrix was then visualized in Prism GraphPad as a heatmap.
RingMapper was run with default parameters and the output file was filtered to include only positive correlations between two G nucleotides. A second filter removed all correlations with Z-scores less than 2.0. Networks of structurally related nucleotides were visualized in Cytoscape with unique G bases represented as nodes and positive correlations represented as edges.
Minimum free energy RNA secondary structure modeling:
The baseline secondary structure model of Spinach was generated in mFold with default parameters. Population average DMS guided RNA secondary structure modeling was performed as described previously in the DANCE pipeline. Briefly, the python script DanceMapper.py was run on parsed mutation output files with the following settings for deriving population average data: --fit --maxc 1. Then, the population average reactivities were converted to free energy restraints for the RNA folding program RNAstructure through the foldClusters.py script. When processing BASH MaP data, the additional command --nog was used with foldClusters.py to ignore the reactivity data for G nucleotides. The resulting RNA secondary structures were visualized in RNA2D drawer. Mapping of per nucleotide DMS reactivity onto RNA2D drawer secondary models was performed through the custom python script color_reactivities_RNA2D_drawer.py. Conformation specific RNA secondary structure modeling was performed by increasing the maximum allowed conformations from 1 to 5 with -- maxc 5.
Single molecule probabilistic RNA secondary structure modeling:
A custom pipeline was developed to apply the DaVinci structural analysis method to BASH MaP56. This analysis method utilizes misincorporations to generate a folding constraint for each sequencing read. Folding constraints are then passed to ContraFold which folds each sequencing read as a unique RNA molecule. The complete analysis pipeline is as follows:
First, sequencing data is processed through ShapeMapper as described above with --amplicon --dms --output-parsed --min-mutation-separation 2 options enabled. The resulting .mut file is then filtered and converted to a .simple file with mut_to_simple.py. For DMS MaP experiments, the resulting .simple file is converted into a .bit file by running the simple_to_bit.py python script. For BASH MaP experiments, the resulting .simple file is instead converted into a .bit file with the script updated_simple_to_bit_rG42.py. This script requires an input fasta file and sets all upper-case G positions to be considered for base pairing. In addition, the script constrains all lowercase letters to be single stranded in the .bit file. If a sequencing read harbors a misincorporations at the position of one of the lowercase letters, then all lowercase letters in that sequencing read are instead considered for base pairing. The resulting .bit file is then passed as an argument to the fold-contrafold-uniq-bits-vectors2.py script. ContraFold was run either with normal settings or with --noncomplementary enabled. Typically, the fold-contrafold-uniq-bits-vectors2.py script is stopped after roughly 10,000 RNA molecules have been folded to save on computation time. The forgi vector encodings of the RNA secondary structures generated by ContraFold are output in the file forgi-vect-ser2.txt. Dimensional reduction is performed on the forgi vectors text file with the python script run-pca-on-forgi-vectors.py which reduces the vector space to 2 dimensions. Kmeans clustering is then performed on the 2-dimensional representation of RNA structures with the draw-kmeans-clusters.py script. Identification of a population average structure is done by choosing --num_clusters 1. Multiple conformational analysis is performed by varying the number of Kmeans clusters. The structure of the most representative of the Kmeans cluster is retrieved with the python script fetch_DaVinci_folded_copy.py and visualized with RNA 2D drawer.
Nucleotide reactivity normalization:
Mutation rates were normalized to reactivity values as previously described with the custom python script normalize_DAGGER_DANCE.py.
Tertiary folding constraint determination and implementation (DAGGER):
G’s likely to be engaged in tertiary interactions were identified by first selecting G’s in the bottom quartile for misincorporation rate. In situations when reactivity data was available for multiple conformations, G’s in separate conformations were treated as unique G’s and all unique G’s were pooled for reactivity normalization. From the bottom quartile of reactive G’s, G’s which displayed correlations to other lowly reactive G’s as identified by RingMapper were designated as engaged in a tertiary interaction.
The DaVinci analysis pipeline was modified to utilize tertiary constraints through setting tertiary G positions to lowercase G’s in the fasta input file for generation of .bit files by updated_simple_to_bit_rG42.py. The modified DaVinci analysis pipeline is reffered to as DAGGER.
All python scripts for analyzing data can be found at: https://github.com/SamieJaffreyLab/DAGGER_MaP
Assembly: custom fasta files for each RNA construct
Supplementary files format and content: Shapemapper output files include mutation rates for each nucelotide as output by ShapeMapper2. A complete description of the ShapeMapper output is available at (https://github.com/Weeks-UNC/shapemapper2). Mutate and MaP experiments processed files include a Z-score normalized mutate and map heatmap. See https://github.com/SamieJaffreyLab/DAGGER_MaP for detailed description of the heatmap file formats.
Library strategy: Amplicon sequencing

growth_protocol: HeLa cells were grown in DMEM containing 10% FBS with 100 U ml−1penicillin and 100 µg ml−1streptomycin under standard culturing conditions (37°C with 5.0% CO2). SH-SY5Y cells were grown in a 1:1 ratio of DMEM to F12 media containing 10% FBS with 100 U ml−1penicillin and 100 µg ml−1streptomycin under standard culturing conditions (37°C with 5.0% CO2).

extract_protocol:
In vitro transcribed RNA was purified from the quenched DMS reaction with RNA Clean and Concentrator 5 kit and eluted in 10µL water. RNA was either directly processed or stored at -80°C
We noticed that DMS probing caused a large portion of adherent cells to become detached from the plate. We therefore decanted the quenched DMS probing media off the plate into a 15 mL falcon tube and pelleted the detached cells for 5 min at 4C and 1,000 x g. To isolate the total RNA, 2 mL Trizol reagent was added to the plate for the cells which had not become detached. Then, the 2 mL Trizol reagent was transferred to solubilize the pelleted cells which had become detached during DMS probing. Total RNA was then isolated according to manufacturer’s instructions.
Sequencing library preparation:
Reverse transcription
Superscript II: Reverse transcription with SSII was performed as follows. 200 ng of input RNA was mixed with 1µL 50µM random hexamer (HeLa total RNA) or 2µL of 1µM gene specific reverse transcription primer (in vitro transcribed RNAs) and annealed. Annealed RNA was added to reverse transcription master mix containing 6 mM final concentration of MnCl2. Reverse transcription reactions were incubated at 23°C for 15 min only when using random hexamers then 42°C for 3 hours.
Marathon RT: Reverse transcription with Marathon RT was performed with a modified protocol. First, 200 ng of input RNA was mixed with 2µL10 mM dNTP and 1µL 50µM random hexamer or 2µL of 1µM gene specific RT primer. The volume was brough up to 8µL and the RNA was annealed to the primer with the following thermocycler settings: 80°C for 1 min, 65°C for 5 min at which point the RNA was placed on ice for 2 min. Then, an RT master mix was created containing 4µL of 5x Marathon MaP buffer (250 mM Tris-HCl pH 7.5, 1 M NaCl), 4µLof 100% glycerol, 1µL of 100 mM DTT, 0.5µL of 20 mM MnCl2, 0.5µL of water. Next, 10µL of RT master mix was added to 8µL of annealed RNA followed by the addition of 2µL Marathon RT. Reverse transcription reactions were incubated at 23°C for 15 min only when using random hexamers then 42°C for 3 hours.
Reverse transcription reactions were inactivated by incubating at 75°C for 10 min. cDNA was purified with DNA clean and concentrator 5 columns by using 7 volumes of DNA binding buffer (140 µL) and eluted in 10µL water.
Sequencing libraries were generated via a 2 step PCR. Briefly, 1/5th of purified cDNA (2 µL) was amplified with step 1 forward and reverse primers. HeLa 18s rRNA samples included a 4-nucleotide barcode on the forward primer and a 7-nucleotide barcode on the reverse primer which enabled pooling of purified step 1 PCR products. AKT2 step 1 PCR primers were designed to amplify a ∼200 nucleotide segment of the AKT2 3’UTR. In vitro transcribed RNAs used non-barcoded step 1 forward and reverse primers which were specific to flanking sequences introduced into the RNA constructs. These flanking sequences are referred to as a Structure Cassette (SC). Step 1 PCR reactions were assembled on ice by adding to 2µL purified cDNA: 1.25µL of 10µM forward primer, 1.25µL of 10µM reverse primer, 9µL of water and 12.5µL of 2x Phusion HF master mix or 2x Phusion GC master mix for AKT2 step 1 PCR. Step 1 PCR reactions were run with the following thermocycler settings: 98°C for 2 min followed by 8-28 cycles of 98°C for 10 seconds, 65°C for 20 seconds, 72°C for 20 seconds, followed by a final extension of 2 min. PCR cycle number was optimized for each experiment separately. PCR reactions were purified with 1.8x AMPure XP beads according to manufactures instructions. Purified DNA was eluted in 25µL water.
Step 2 PCR was performed with NEB Next Multiplex Oligos for Illumina. Step 2 PCR reactions were composed of the following: 2-10 ng of purified step 1 DNA brough up to 12µL water, 4µL of i5 index primer or universal primer, 4µL i7 index primer, and 20µL of 2x Phusion HF or 2x Phusion GC for AKT2 library prep. Step 2 PCR reactions were run with the following thermocycler settings: 98°C for 2 min followed by 6-12 cycles of 98°C for 15 seconds, 65°C for 30 seconds, 72°C for 30 seconds, then 72°C for 5 min. PCR cycle number was optimized for each experiment separately. Step 2 PCR reactions were size selected with AMPure XP beads, eluted in 25µL of water, and quantified with Qubit 1x dsDNA HS kit. Libraries were pooled and sequenced on the NovaSeq 6000 PE 2x100, NovaSeq 6000 PE 2x150, MiSeq PE 2x150, Nextseq2000 P1 600 cycles, or MiSeq Micro 300 cycles.
Gene-specific BASH MaP:
Total RNA was treated with DNAse I for 30 min prior to BASH MaP treatment according to manufacturer’s instructions. Three replicates of 1 µg of total RNA was mixed with 1µL 50µM random hexamers and the volume was adjusted to 11 µL. RNA was annealed by heating to 85°C for 1 min, 65°C for 5 min, then placing on ice for 3 min. Reverse transcription was performed with SuperScript II as described previously8. Reverse transcription reactions were incubated at 23°C for 15 min and then 42°C for 3 hours. Reactions were inactivated by heating to 75°C for 10 min. Reverse transcription replicates were pooled and the cDNA was purified with the Zymo DNA Clean and Concentrator 5 kit and eluted in 10µL water.

treatment_protocol:
DMS modification of in vitro transcribed RNA
In vitro transcribed RNA was probed as follows. 200 ng – 1 µg of RNA was folded in a buffer designed to promote modification of A and C bases only (200 mM Bicine pH 7.75 at room temperature, 100 mM KCl) or a buffer which promotes the additional modification of m1G and m3U (200 mM Bicine pH 8.37 at room temperature, 100 mM KCl). RNA was refolded in buffer by heating to 95°C for 3 min then snap cooling on ice for 1 min. Then MgCl2 was added to a final concentration of 1-5 mM and the RNA was left at room temperature for 10 min. Neat DMS was diluted to 1.7M with anhydrous ethanol and added to the folded RNA. DMS reactions was performed for 10 min at room temperature or 6 min at 37°C. Spinach was probed at room temperature due to its thermal instability. pUG RNA was probed at 37°C. Reactions were quenched by the addition of 4 volumes of 20% beta-mercaptoethanol in water on ice. RNA was purified from the quenched DMS reaction with RNA Clean and Concentrator 5 kit and eluted in 10µL water. RNA was either directly processed or stored at -80°C.
In cell DMS modification and purification
DMS modification of SH-SY5Y cells was performed as follows. Cells were grown to 85% confluence in a 10 cm dish. Then, the media was exchanged for a DMS probing media which consisted of 1:1 DMEM to F12 medium with 10% FBS and 200 mM Bicine adjusted to pH 8.37 with NaOH at room temperature. Probing media was prewarmed to 37C and cell media was exchanged with 5.4 mL probing media for 3 min at 37°C. Then 600µL of prewarmed 1.7 M DMS or prewarmed 600µL ethanol was added to the cells and incubated for 6 min at 37°C. DMS reaction was stopped by the addition of 6 mL 20% 2-mercaptoethanol and cells were placed on ice. We noticed that DMS probing caused a large portion of adherent cells to become detached from the plate. We therefore decanted the quenched DMS probing media off the plate into a 15 mL falcon tube and pelleted the detached cells for 5 min at 4C and 1,000 x g. To isolate the total RNA, 2 mL Trizol reagent was added to the plate for the cells which had not become detached. Then, the 2 mL Trizol reagent was transferred to solubilize the pelleted cells which had become detached during DMS probing. Total RNA was then isolated according to manufacturer’s instructions.
M2 BASH MaP
Spinach and polyUG T7 templates were randomly mutagenized by performing 24 rounds of error-prone PCR. Mutagenized Spinach and polyUG were then in vitro transcribed, folded and DMS modified as described prior with minor modifications. MgCl2 was added to a final concentration of 5 mM and Spinach was probed in the presence of 5µM DFHBI-1T. polyUG RNA was probed in the presence of 2µM NMM.

value: neuroblastoma

value: WT

value: EtOH no DMS control


title: AKT2, BASH MaP, 170 mM, SSII

description:
Duration: 6 min ;temperature: 37C;buffer: SH-SY5Y, 200 mM Bicine pH 8.3, ;ligand: N/A
AKT2 DAGGER MaP

value: DMS followed by BASH


title: D5-Teff-Rest-MED12-KO-Input_ChIP

value: Rest


title: D5-Teff-Stim-MED12-KO-Input_ChIP


title: D5-Teff-Rest-AAVS1-KO-Input_ChIP


title: D4-Teff-Rest-AAVS1-KO-Input_ChIP


title: D4-Teff-Stim-MED12-KO-Input_ChIP


title: SFSWAP knockdown - Biological Replicate 2  (TG-treated)


title: Non-target - Biological Replicate 3


title: D4-Teff-Stim-MED12-KO-MED12Thermo_ChIP

value: MED12Thermo (Bethyl/Thermo A300-774A)


title: D5-Teff-Rest-MED12-KO-MED12Thermo_ChIP


title: D4-Teff-Rest-AAVS1-KO-MED12Thermo_ChIP


title: D4-Teff-Rest-MED12-KO-MED12Thermo_ChIP


title: D4-Teff-Rest-AAVS1-KO-PolPS2_ChIP

value: PolPS2 (Abcam ab5095)


title: D5-Teff-Stim-MED12-KO-MED12Thermo_ChIP


title: D4-Teff-Rest-AAVS1-KO-CXXC1_ChIP

value: CXXC1 (Cell Signaling 40672S)


title: D4-Teff-Rest-MED12-KO-NELFA_ChIP

value: NELFA (Santa Cruz sc-365004)


title: D4-Teff-Rest-MED12-KO-PolPS5_ChIP

value: PolPS5 (Abcam ab5131)


title: D4-Teff-Rest-AAVS1-KO-PolPS5_ChIP


title: D4-Teff-Stim-AAVS1-KO-CXXC1_ChIP


title: D4-Teff-Rest-MED12-KO-PolCTD_ChIP

value: PolCTD (Cell Signaling 2629)


title: D4-Teff-Rest-MED12-KO-Input_ChIP


title: D4-Teff-Rest-MED12-KO-PolPS2_ChIP


title: D4-Teff-Stim-AAVS1-KO-NELFA_ChIP


title: D4-Teff-Rest-AAVS1-KO-PolCTD_ChIP


title: D4-Teff-Stim-AAVS1-KO-MED12Thermo_ChIP


title: D4-Teff-Stim-AAVS1-KO-PolPS5_ChIP


title: D4-Teff-Stim-AAVS1-KO-PolPS2_ChIP


title: D4-Teff-Stim-MED12-KO-PolPS2_ChIP


title: D4-Teff-Stim-MED12-KO-PolCTD_ChIP


title: D5-Teff-Rest-AAVS1-KO-CXXC1_ChIP


title: D4-Teff-Stim-MED12-KO-NELFA_ChIP


title: D5-Teff-Rest-AAVS1-KO-MED12Thermo_ChIP


title: D4-Teff-Rest-AAVS1-KO-NELFA_ChIP


title: D5-Teff-Rest-AAVS1-KO-PolPS2_ChIP


title: D5-Teff-Rest-AAVS1-KO-PolCTD_ChIP


title: D5-Teff-Rest-AAVS1-KO-PolPS5_ChIP


title: D5-Teff-Rest-MED12-KO-NELFA_ChIP


title: D5-Teff-Rest-MED12-KO-PolCTD_ChIP


title: D5-Teff-Rest-MED12-KO-PolPS2_ChIP


title: D5-Teff-Rest-AAVS1-KO-NELFA_ChIP


title: D4-Teff-Stim-AAVS1-KO-PolCTD_ChIP


title: D5-Teff-Stim-AAVS1-KO-CXXC1_ChIP


title: D5-Teff-Rest-MED12-KO-PolPS5_ChIP


title: D5-Teff-Stim-AAVS1-KO-PolCTD_ChIP


title: D5-Teff-Stim-AAVS1-KO-MED12Thermo_ChIP


title: D5-Teff-Stim-MED12-KO-PolPS2_ChIP


title: D5-Teff-Stim-MED12-KO-PolCTD_ChIP


title: D5-Teff-Stim-AAVS1-KO-NELFA_ChIP


title: D5-Teff-Stim-AAVS1-KO-PolPS5_ChIP


title: D4-Teff-Stim-MED12-KO-PolPS5_ChIP


title: D5-Teff-Stim-AAVS1-KO-PolPS2_ChIP


title: D5-Teff-Stim-MED12-KO-NELFA_ChIP


title: D5-Teff-Stim-MED12-KO-PolPS5_ChIP


title: D6-Teff-Stim-SELA-H3K4me3_CUT&RUN

data_processing:
Samples were processed according to Zheng et al. (dx.doi.org/10.17504/protocols.io.bjk2kkye)
Bcl2fastq (v2.19) with the settings --minimum-trimmed-read-length 8 was used to generate fastqs.The fastqs were trimmed with cutadapt (v1.18)
Bowtie2 (v2.2.5) was used to align the trimmed fastqs to GRCh38 using settings --local --very-sensitive --no-mixed --no-discordant --phred33 --dovetail -I 10 -X 700 -p 8 -q and E. coli (EMBL accession U00096.2) with settings --local --very-sensitive --no-overlap --no-dovetail --no-mixed --no-discordant --phred33 -I 10 -X 700 -p 8 -q.
Bam files were generated with samtools(version 1.9) view -bS -F 0x04 and bam to bed conversion performed with bedtools (v2.30.0) bamtobed -bedpe.
Bedfiles were filtered to include only paired reads of less than 1000 bp with the command awk '$1==$4 && $6-$2 < 1000 {print $0}' samplename.bed before generating spikein normalized bedgraph files using bedtools (version 2.30.0) genomecov -bg and a scale factor calculated as scale_factor=`echo "10000 / $seqDepth" | bc -l`.
Peak calling was performed using spikein normalized bedgraph files as input with SEACR (v1.3). For each donor and conditon with a corresponding IgG file, the IgG file was used to determine the background using the non and stringent options. The top 1% of peaks for each sample were defined using the non and stringent options.
ChIP-seq blacklist regions were removed from filtered SEACR files prior to generating the sample by peak matrix
Scaling of bedgraphs was performed using DeSeq2 with regions present in the peak by sample matrix generated from AAVS1 KO and MED12 KO samples
Assembly: GRCh38
Supplementary files format and content: Scaled bedgraphs are provided for methylation samples and unscaled bedgraphs for IgG control samples
Supplementary files format and content: SEACR peaks are provided for methylation samples
Library strategy: CUT&RUN

growth_protocol: CD4+ effector T cells were isolated from fresh Peripheral Blood Leukopaks (STEMCELL Technologies, #70500) from healthy human donors, after institutional review board–approved informed written consent (STEMCELL Technologies). The contents of the Leukopaks were washed twice with a 1X volume of EasySep buffer (DPBS, 2% fetal Bovine Serum (FBS), 1mM pH 8.0 EDTA) using centrifugation. The washed cells were resuspended at 200E6 cells/mL in EasySep buffer and isolated with the EasySep™ Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit (STEMCELL Technologies, #18063), according to the manufacturer’s protocol. Teffs were seeded at 1x106 cells/mL in RPMI-1640 supplemented with 10% FCS, 2 mM L-Glutamine (Fisher Scientific #25030081), 10 mM HEPES (Sigma, #H0887-100ML), 1X MEM Non-essential Amino Acids (Fisher, #11140050), 1 mM Sodium Pyruvate (Fisher Scientific #11360070), 100 U/mL Penicillin-Streptomycin (Sigma, #P4333-100ML), and 50 U/mL IL-2 (Amerisource Bergen, #10101641). Cells were then stimulated with ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator (STEMCELL Technologies, #10990) at 6.25 uL/mL for Teff. Following activation and electroporation, cells were split 1:2 every 48 hours to maintain an approximate density of 1x106 cells/mL and supplemented with IL-2.

extract_protocol:
CUT&RUN from resting and 48-hour restimulated cells was performed according to the manufacturer’s protocol with the EpiCypher CUTANA™ ChIC/CUT&RUN Kit and provided reagents. In brief, 5E5 T cells per reaction were washed with PBS before nuclear isolation using the EpiCypher recommended lysis buffer consisting of 20 mM HEPES pH 7.9 (Sigma-Aldrich), 10 mM KCl (Sigma-Aldrich), 0.1% Triton X-100 (Sigma-Aldrich), 20% Glycerol (Sigma-Aldrich), 1 mM MnCl2 (Sigma-Aldrich), 1X cOmplete Mini-Tablet (Roche, # 11873580001), and 0.5 mM Spermidine (Sigma-Aldrich). The cells were resuspended in 100 µL per reaction cold nuclear extraction buffer and incubated on ice for 10 minutes. Following lysis, nuclei were pelleted and resuspended in 100 µL per reaction of nuclear extraction buffer. The isolated nuclei were then frozen at -80 in extraction buffer until DNA isolation. After thawing the samples at 37C, the nuclei were bound to activated conA beads. After adsorption of nuclei to beads, permeabilization was performed with 0.01% digitonin containing buffer. Antibodies for H3K4me1 (EpiCypher, #13-0057), H3K4me2 (EpiCypher, #13-0027), H3K4me3 (EpiCypher, #13-0041) and IgG (EpiCypher, #13-0042) were added at 500 ng per reaction. Following overnight antibody binding, pAG-MNase addition, and chromatin cleavage, 0.5 ng of the provided E. coli DNA was added to each sample following chromatin cleavage by MNase. The provided spin columns and buffers were used for DNA isolation and purification.
The resulting DNA was prepared for sequencing using the CUTANA™ CUT&RUN Library Prep Kit (Cat 14-1002) according to the manufacturer’s protocol.

treatment_protocol: Guide loaded Cas9 RNPs were assembled with custom crRNAs (Dharmacon) which were resuspended in IDT duplex buffer (IDT, #11-01-03-01) at 160uM. Dharmacon Edit-R CRISPR-Cas9 Synthetic tracrRNA (Dharmacon, #U-002005-20) also resuspended in Nuclease Free Duplex Buffer at 160uM was combined at a 1:1 molar ratio in a 96 well plate and incubated at 37°C for 30 minutes. Single-stranded donor oligonucleotides (ssODN; sequence: TTAGCTCTGTTTACGTCCCAGCGGGCATGAGAGTAACAAGAGGGTGTGGTAATATTACGGTACCGAGCACTATCGATACAATATGTGTCATACGGACACG, 100uM stock) was added to the complex at a 1:1 molar ratio and incubated at 37°C for 5 minutes. Finally, Cas9 protein (MacroLab, Berkeley, 40 µM stock) was added at a 1:2 molar ratio and incubated at 37°C for 15 minutes. The resulting RNPs were frozen at -80°C until the day of electroporation and were thawed to room temperature prior to use. 48 hours following T cell activation, the cells were pelleted at 100x g for 10 minutes and resuspended in room temperature P3 Primary Cell Nucleofector Buffer (Lonza, catalog no. V4XP-3032) at 1.5E6 cells per 17.8 µL. 1.5E6 cells were transferred to each RNP containing well and mixed gently. 25 µL of the combined RNP cell solution was transferred to a 96-well electroporation cuvette plate (Lonza, #VVPA-1002) and nucleofected with pulse code DS-137. Immediately following electroporation, the cells were gently resuspended in 90 µL warmed media and incubated at 37 C for 15 minutes. After recovery, the cells were cultured in 96 well round-bottom plates at 1x106 cells/mL for the duration of the experiment. To prevent edge effects, the guides were randomly distributed across each plate and the first and last columns and rows of each plate was filled with PBS to prevent evaporation. CRISPR-Cas9 edited cells were restimulated on day 8 following isolation for stimulation response arrayed assays with ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator (STEMCELL Technologies, #10990). Kinase inhibitor SEL120-34A studies were performed with unedited cells cultured as described above. SEL120-34A (Selleckchem, #S8840) was reconstituted in ultrapure H2O according to the manufacturer’s recommendations. Cells were treated every 48 hours with a 1 uM dose and treatment was started 48 hours following cell isolation to align with the time at which cells are edited in CRISPR based experiments. Restimulation of cells for flow cytometry and CUT&RUN was performed 10 days after initial isolation.

tag: expt

value: SELA

tag: group


title: D6-Teff-Stim-SELA-IgG_CUT&RUN

value: IgG


title: D6-Teff-Stim-H2O-IgG_CUT&RUN

value: Blood


title: D6-Teff-Stim-H2O-H3K4me3_CUT&RUN


title: D6-Teff-Stim-H2O-H3K4me1_CUT&RUN


title: D6-Teff-Stim-SELA-H3K4me2_CUT&RUN


title: D6-Teff-Stim-SELA-H3K4me1_CUT&RUN


title: D3-Teff-Stim-MED12-KO-H3K4me2_CUT&RUN

value: CRISPR KO


title: D3-Teff-Stim-MED12-KO-H3K4me1_CUT&RUN

value: CD4+ Teff


title: D3-Teff-Stim-MED12-KO-H3K4me3_CUT&RUN

value: Stim


title: D3-Teff-Stim-AAVS1-KO-H3K4me2_CUT&RUN


title: D6-Teff-Resting-H2O-H3K4me3_CUT&RUN

value: Resting


title: D6-Teff-Resting-H2O-H3K4me1_CUT&RUN


title: D3-Teff-Stim-AAVS1-KO-H3K4me1_CUT&RUN


title: D6-Teff-Resting-SELA-H3K4me1_CUT&RUN


title: D6-Teff-Resting-H2O-H3K4me2_CUT&RUN


title: D6-Teff-Stim-H2O-H3K4me2_CUT&RUN


title: D3-Teff-Resting-MED12-KO-H3K4me1_CUT&RUN


title: D3-Teff-Resting-AAVS1-KO-H3K4me2_CUT&RUN


title: D3-Teff-Resting-AAVS1-KO-H3K4me1_CUT&RUN


title: D5-Teff-Stim-H2O-IgG_CUT&RUN


title: D3-Teff-Resting-AAVS1-KO-H3K4me3_CUT&RUN


title: D3-Teff-Resting-MED12-KO-H3K4me2_CUT&RUN


title: D3-Teff-Resting-MED12-KO-H3K4me3_CUT&RUN


title: D3-Teff-Resting-AAVS1-KO-IgG_CUT&RUN


title: D5-Teff-Stim-H2O-H3K4me3_CUT&RUN


title: D5-Teff-Stim-H2O-H3K4me2_CUT&RUN


title: D5-Teff-Stim-SELA-IgG_CUT&RUN


title: D3-Teff-Stim-AAVS1-KO-H3K4me3_CUT&RUN


title: D3-Teff-Stim-AAVS1-KO-IgG_CUT&RUN


title: D5-Teff-Stim-SELA-H3K4me2_CUT&RUN


title: D6-Teff-Resting-SELA-IgG_CUT&RUN


title: D6-Teff-Resting-H2O-IgG_CUT&RUN


title: D6-Teff-Resting-SELA-H3K4me3_CUT&RUN


title: D2-Teff-Stim-MED12-KO-H3K4me3_CUT&RUN


title: D2-Teff-Stim-MED12-KO-IgG_CUT&RUN


title: D2-Teff-Stim-AAVS1-KO-IgG_CUT&RUN


title: D3-Teff-Resting-MED12-KO-IgG_CUT&RUN


title: D2-Teff-Stim-MED12-KO-H3K4me2_CUT&RUN


title: D2-Teff-Stim-AAVS1-KO-H3K4me3_CUT&RUN


title: D2-Teff-Stim-AAVS1-KO-H3K4me2_CUT&RUN


title: D2-Teff-Stim-MED12-KO-H3K4me1_CUT&RUN


title: D5-Teff-Resting-H2O-H3K4me2_CUT&RUN


title: D2-Teff-Stim-AAVS1-KO-H3K4me1_CUT&RUN


title: D5-Teff-Resting-H2O-IgG_CUT&RUN


title: D5-Teff-Resting-SELA-H3K4me3_CUT&RUN


title: D5-Teff-Resting-SELA-IgG_CUT&RUN


title: SMC Donor4, Proliferative, H3K4me3

value: Ascending Aorta

value: Smooth Muscle Cell

value: female

value: Proliferative


title: D5-Teff-Resting-H2O-H3K4me3_CUT&RUN


title: D5-Teff-Resting-SELA-H3K4me2_CUT&RUN


title: D5-Teff-Resting-SELA-H3K4me1_CUT&RUN


title: D2-Teff-Resting-MED12-KO-H3K4me3_CUT&RUN


title: D2-Teff-Resting-MED12-KO-IgG_CUT&RUN


title: D2-Teff-Resting-MED12-KO-H3K4me2_CUT&RUN


title: SMC Donor5, Proliferative, H3K27ac


title: D2-Teff-Resting-MED12-KO-H3K4me1_CUT&RUN


title: D2-Teff-Resting-AAVS1-KO-IgG_CUT&RUN


title: D2-Teff-Resting-AAVS1-KO-H3K4me3_CUT&RUN


title: D2-Teff-Resting-AAVS1-KO-H3K4me1_CUT&RUN


title: D4-Teff-Stim-H2O-H3K4me3_CUT&RUN


title: D2-Teff-Resting-AAVS1-KO-H3K4me2_CUT&RUN


title: D4-Teff-Stim-H2O-H3K4me2_CUT&RUN


title: D4-Teff-Stim-SELA-H3K4me3_CUT&RUN


title: D4-Teff-Stim-H2O-H3K4me1_CUT&RUN


title: D1-Teff-Stim-MED12-KO-IgG_CUT&RUN


title: D4-Teff-Stim-SELA-IgG_CUT&RUN


title: D3-Teff-Stim-MED12-KO-IgG_CUT&RUN


title: D1-Teff-Stim-MED12-KO-H3K4me1_CUT&RUN


title: D1-Teff-Stim-AAVS1-KO-H3K4me3_CUT&RUN


title: D1-Teff-Stim-AAVS1-KO-IgG_CUT&RUN


title: D1-Teff-Stim-MED12-KO-H3K4me2_CUT&RUN


title: D4-Teff-Stim-SELA-H3K4me2_CUT&RUN


title: D1-Teff-Stim-MED12-KO-H3K4me3_CUT&RUN


title: D1-Teff-Stim-AAVS1-KO-H3K4me1_CUT&RUN


title: D4-Teff-Resting-H2O-IgG_CUT&RUN


title: D1-Teff-Stim-AAVS1-KO-H3K4me2_CUT&RUN


title: D4-Teff-Resting-H2O-H3K4me3_CUT&RUN


title: D4-Teff-Resting-H2O-H3K4me2_CUT&RUN


title: D4-Teff-Resting-SELA-IgG_CUT&RUN


title: D4-Teff-Resting-H2O-H3K4me1_CUT&RUN


title: D4-Teff-Stim-SELA-H3K4me1_CUT&RUN


title: D4-Teff-Resting-SELA-H3K4me2_CUT&RUN


title: D4-Teff-Resting-SELA-H3K4me1_CUT&RUN


title: D1-Teff-Resting-MED12-KO-H3K4me2_CUT&RUN


title: D1-Teff-Resting-MED12-KO-H3K4me1_CUT&RUN


title: D1-Teff-Resting-MED12-KO-H3K4me3_CUT&RUN


title: D1-Teff-Resting-AAVS1-KO-IgG_CUT&RUN


title: D1-Teff-Resting-MED12-KO-IgG_CUT&RUN


title: D1-Teff-Resting-AAVS1-KO-H3K4me2_CUT&RUN


title: D1-Teff-Resting-AAVS1-KO-H3K4me3_CUT&RUN


title: PFOS_3

city: Lanzhou

first: Haofei

last: Shen

address: No.1 Donggang West Road, Chengguan District

country: China

institute: Lanzhou University

data_processing:
Remove the adapter sequences in the reads, remove the reads that have no inserted fragments due to adapter self-linkage, etc.; trim off the low quality (quality value less than 20) bases at the end of the sequence (3' end), if there are any quality value less than 10 in the remaining sequences, then the whole sequence will be eliminated; otherwise, retain it; and remove reads that contain N-ratio of more than 10%; Remove the reads with N-ratio more than 10%; discard the sequences with length less than 20bp after adapter and quality trimming. Software used: fastp(https://github.com/OpenGene/fastp)
The clean data (reads) after quality control were compared with the reference genome to obtain mapped data (reads) for subsequent transcript assembly, expression calculation, etc. At the same time, the quality of the comparison results of the transcriptome sequencing was evaluated using the software: HiSat2 (http://ccb.jhu.edu/). software/hisat2/index.shtml).
The software used for differential expression was DESeq2 (with biological replicates, used by default) or DEGseq (without biological replicates, used by default), and the default screening criteria for significantly differentially expressed genes were: FDR<0.05 & |log2FC|≧ 1. When a gene satisfies both of these two conditions at the same time, it is regarded as a differentially expressed gene (DEG) or differentially expressed gene (DEG).
The expression levels of genes and transcripts were quantitatively analyzed using the software RSEM to facilitate the subsequent analysis of gene/transcript differential expression among different samples and to reveal the regulatory mechanisms of genes by combining the functional information of the sequences.
Assembly: hg38
Supplementary files format and content: gene.count.matrix.annot.xlsx

source_name: Primary human endometrial mesenchymal cells

extract_protocol:
Total RNA was extracted from the tissue samples, and the concentration and purity of the extracted RNA were detected by Nanodrop2000, RNA integrity was detected by agarose gel electrophoresis, and RQN values were determined by Agilent5300. The requirements for a single library were 1 μg of total RNA, concentration ≥ 30 ng/μL, RQN > 6.5 and OD260/280 between 1.8 and 2.2.
① Extraction of total RNA ② Oligo dT enrichment of mRNA ③ Fragmentation of mRNA ④ Reverse synthesis of cDNA ⑤ Ligation of adapter ⑥ Fragmentation screening and library enrichment ⑦ Sequencing on the NovaSeqX plus platform


title: Control_3

value: Primary human endometrial mesenchymal cells


title: Control_2


title: Control_1


title: PFOS_2


title: PFOS_1


title: C57/BL6, 6mo Smoke, 6

city: Pittsburgh

first: Justin

last: Sui

state: PA

address: 200 Lothrop Street

institute: University of Pittsburgh School of Medicine

data_processing:
Raw data were demultiplexed using Cell Ranger 5.0.1 and the mkfastq function and then aligned to 10X Genomics’ mouse reference genome mm10-2020-A using cellranger count.
Assembly: mm10-2020-A
Supplementary files format and content: processed files contained outputs from CellRanger including barcodes, features, and matrix

organism: Mus musculus

source_name: whole lung

extract_protocol:
left lung lobe from each mouse was further processed to isolate single cell suspensions. Lung tissue was first minced into small pieces and placed in 0.5mL Dispase solution (50U/mL, Sigma) in a conical tube. The tissue was incubated at room temperature on a gentle rocker for 45 minutes. The tissue was then placed in a petri dish with 7mL complete media with 10ml DNase-I solution (1mg/mL, Sigma), teased apart, and placed on a rocker for 12 minutes to liberate adherent cells. Liberated cells were then passed through a 70mm strainer into a conical tube with subsequent washing of the strainer with 5mL of media. This process was repeated with a 40mm strainer. Cells were spun down at 400g for 10minutes at 4 degrees. Cells were resuspended in 6mL of red blood cell lysis buffer, incubated for 5 minutes, and spun down.  Cell pellets were resuspended in 0.5mL PBS with 0.4% bovine serum albumin (BSA, Albuminx from ThermoFisher), filtered a final time through a 40mm FloMi filter tip (Bel-Art), and kept on ice. Cells were counted and 5,000 cells were processed. Individual cells were barcoded with library preparation using 3primeV2 reagents by 10X Genomics Chromium System per the manufacturer’s protocol.
Next generation RNA sequencing was performed on the libraries using an Illumina NextSeq-500 by the UPMC Genome Center (Pittsburgh, PA).

value: whole lung

tag: age

value: 10-12wks age

tag: strain

value: C57/BL6 Jackson

value: Smoke 4 cigarettes/day 6 months tx


title: C57/BL6, 6mo Smoke, 8


title: C57/BL6, 6mo Smoke, 5


title: D4-Teff-Resting-SELA-H3K4me3_CUT&RUN


title: C57/BL6, 6mo Air, 4

value: room air 6 months tx


title: C57/BL6, 6mo Air, 3


title: C57/BL6, 6mo Air, 2


title: D5-Teff-Resting-H2O-H3K4me1_CUT&RUN

value: SELA


title: D1-Teff-Resting-AAVS1-KO-H3K4me1_CUT&RUN


title: RNA-seq_Sox2_KO_LateICM_2-10

city: Muenster

first: Yanlin

last: Hou

address: Röntgenstrasse 20

country: Germany

institute: Max Planck Institute for Molecular Biomedicine

data_processing:
for RNA-seq, UMI sequence of each read was extracted from FASTQ files for Read 2 using UMI-tools. Poly-A sequences were trimmed from the 3' end of Read 1 using Cutadapt (https://cutadapt.readthedocs.org/). The trimmed Read 1 sequences were aligned to the mm10 mouse reference genome using TopHat2 with GENCODE (vM23) transcripts as a transcriptome reference and with default parameters. The reads were also mapped to ERCC reference sequences using Bowtie2 with default parameters. To confirm the genotype of Oct4 and Sox2 KO embryos, we also mapped the reads to the PGK-pacDtk-P2A-mKO2-hGHpA or PGK-EGFP-hGHpA cassette. The reads mapped to the genome were assigned to GENCODE transcripts using featureCounts. The number of unique transcripts assigned to each GENCODE gene was calculated using TRUmiCount.  The transcript counts were normalized and transformed to log2 scale using the rlog function of the Bioconductor DESeq2 package. Differential expression analysis was performed on genes that were detected in at least half of the samples in at least one stage using DESeq2 and differentially expressed genes were identified with an adjusted p-value <0.05 and log2 fold change ≥1.
Assembly: mm10
Supplementary files format and content: bigwig
Supplementary files format and content: read count

source_name: E4.5 late ICM

extract_protocol:
All the embryos for RNA-seq were collected from female mice superovulated with PMSG and hCG. Early and late morula samples were collected at E2.75 and E3.25, respectively. Early and late ICM samples were collected at E2.5 and cultured in KSOM with MEKi (PD0325901, 1 μM) until early and late blastocyst stages, respectively. ICM were collected by immunosurgery.
Single-embryo RNA-seq was performed as previously described with some modifications (Kurimoto et al. 2007, Nakamura et al. 2015). 2,493 copies of ERCC RNA Spike-In (Ambion) were added to each single-embryo sample. To remove PCR duplicates, we used R2SP-UMI-d(T)24 primer, instead of V1d(T)24 primer, for reverse transcription (RT). To reduce byproducts derived from the RT primer, the poly(A) tailing reaction was performed only for 1 min at 37°C. cDNA was amplified with V3d(T)24 and R2SP primers and Terra PCR Direct Polymerase (Clontech) for 16 cycles. The cDNA was further amplified for four cycles with NH2-V3d(T)24 and NH2-R2SP primers, and purified thrice with 0.6x AMPure XP beads. The 3’ end enriched libraries were constructed using KAPA HyperPlus Library Preparation Kit (VWR International, KK8513) with 6 cycles of PCR. All the oligos used in the RT and library amplification are in Table EV3. The sequencing was performed on the NextSeq 500 system with single-end 75bp.

value: E4.5 late ICM

value: 1 μm PD0325901, from E2.5 to ICM isolation


title: C57/BL6, 6mo Air, 1


title: RNA-seq_Sox2_KO_LateICM_2-8


title: RNA-seq_Sox2_KO_LateICM_2-3


title: RNA-seq_Sox2_KO_LateICM_1-6


title: RNA-seq_Sox2_KO_LateICM_2-6


title: C57/BL6, 6mo Smoke, 7


title: RNA-seq_Sox2_Ctrl_LateICM_2-6


title: RNA-seq_Sox2_KO_LateICM_1-3


title: RNA-seq_Sox2_KO_LateICM_1-1


title: D6-Teff-Resting-SELA-H3K4me2_CUT&RUN

value: Blood


title: RNA-seq_Sox2_Ctrl_LateICM_2-4


title: RNA-seq_Sox2_Ctrl_LateICM_1-10


title: RNA-seq_Sox2_Ctrl_LateICM_1-5


title: RNA-seq_Sox2_Ctrl_LateICM_1-4


title: RNA-seq_Sox2_Ctrl_LateICM_2-3


title: RNA-seq_Sox2_Ctrl_LateICM_1-1


title: RNA-seq_Sox2_Ctrl_LateICM_2-1


title: RNA-seq_Sox2_KO_EarlyICM_11

source_name: E3.75 early ICM

value: E3.75 early ICM


title: RNA-seq_Sox2_KO_EarlyICM_17


title: RNA-seq_Sox2_KO_EarlyICM_21


title: RNA-seq_Sox2_KO_EarlyICM_10


title: RNA-seq_Sox2_KO_EarlyICM_8


title: RNA-seq_Sox2_KO_EarlyICM_2


title: RNA-seq_Sox2_KO_LateICM_1-5


title: RNA-seq_Sox2_KO_EarlyICM_5


title: RNA-seq_Sox2_Ctrl_EarlyICM_11


title: RNA-seq_Sox2_KO_EarlyICM_1


title: RNA-seq_Sox2_Ctrl_EarlyICM_9


title: RNA-seq_Sox2_Ctrl_EarlyICM_15


title: RNA-seq_Sox2_Ctrl_EarlyICM_7


title: RNA-seqSox2_Ctrl_MWT_EarlyICM_2


title: RNA-seqSox2_Ctrl_MWT_EarlyICM_8


title: RNA-seqSox2_Ctrl_MWT_EarlyICM_10


title: RNA-seq_Sox2_Ctrl_EarlyICM_2


title: RNA-seq_Sox2_Ctrl_EarlyICM_4


title: RNA-seqSox2_Ctrl_MWT_EarlyICM_1


title: RNA-seqSox2_Ctrl_MWT_EarlyICM_15


title: RNA-seqSox2_Ctrl_MWT_EarlyICM_14


title: RNA-seq_Sox2_KO_Morula_15

source_name: E2.75 morula

value: E2.75 morula


title: RNA-seq_Sox2_KO_Morula_6


title: RNA-seq_Sox2_Ctrl_EarlyICM_5


title: RNA-seq_Sox2_KO_Morula_21


title: RNA-seq_Sox2_Ctrl_EarlyICM_10


title: RNA-seq_Sox2_KO_Morula_19


title: RNA-seq_Sox2_Ctrl_Morula_20


title: RNA-seq_Sox2_KO_Morula_9


title: RNA-seq_Sox2_KO_Morula_2


title: RNA-seq_Sox2_KO_Morula_3


title: RNA-seq_Sox2_Ctrl_Morula_17


title: RNA-seq_Sox2_Ctrl_Morula_16


title: RNA-seqSox2_Ctrl_MWT_EarlyICM_9


title: RNA-seq_Sox2_Ctrl_Morula_14


title: RNA-seq_Sox2_Ctrl_Morula_5


title: RNA-seq_Sox2_Ctrl_Morula_7


title: RNA-seq_Sox2_Ctrl_Morula_12


title: RNA-seq_Sox2_Ctrl_Morula_1


title: RNA-seq_Oct4_KO_LateICM_2-6


title: RNA-seq_Oct4_KO_LateICM_2-7


title: RNA-seq_Oct4_KO_LateICM_1-8


title: RNA-seq_Oct4_KO_LateICM_2-2


title: RNA-seq_Sox2_KO_Morula_4


title: RNA-seq_Oct4_KO_LateICM_2-5


title: RNA-seq_Oct4_KO_LateICM_1-5


title: RNA-seq_Oct4_KO_LateICM_1-2


title: D5-Teff-Stim-SELA-H3K4me3_CUT&RUN

value: CD4+ Teff

value: Stim


title: RNA-seq_Oct4_KO_LateICM_1-7


title: RNA-seq_Oct4_Ctrl_LateICM_2-9


title: RNA-seq_Oct4_Ctrl_LateICM_2-1


title: D5-Teff-Stim-SELA-H3K4me1_CUT&RUN


title: RNA-seq_Oct4_Ctrl_LateICM_2-4


title: RNA-seq_Oct4_Ctrl_LateICM_1-9


title: RNA-seq_Oct4_Ctrl_LateICM_1-6


title: RNA-seq_Oct4_Ctrl_LateICM_1-2


title: D5-Teff-Stim-H2O-H3K4me1_CUT&RUN


title: RNA-seq_Oct4_Ctrl_LateICM_1-4


title: RNA-seqOct4_Ctrl_MWT_LateICM_20


title: RNA-seqOct4_Ctrl_MWT_LateICM_19


title: RNA-seq_Oct4_Ctrl_LateICM_1-3


title: RNA-seqOct4_Ctrl_MWT_LateICM_15


title: RNA-seqOct4_Ctrl_MWT_LateICM_10


title: RNA-seqOct4_Ctrl_MWT_LateICM_11


title: RNA-seqOct4_Ctrl_MWT_LateICM_7


title: RNA-seq_Oct4_KO_EarlyICM_17


title: RNA-seq_Oct4_KO_EarlyICM_18


title: RNA-seq_Oct4_KO_EarlyICM_10


title: RNA-seq_Oct4_Ctrl_EarlyICM_8


title: RNA-seq_Oct4_Ctrl_EarlyICM_4


title: RNA-seq_Oct4_Ctrl_EarlyICM_3


title: RNA-seq_Oct4_KO_EarlyICM_1


title: RNA-seq_Oct4_Ctrl_EarlyICM_7


title: RNA-seq_Oct4_Ctrl_EarlyICM_1


title: RNA-seq_Oct4_KO_EarlyICM_2


title: RNA-seq_Oct4_Ctrl_EarlyICM_2


title: RNA-seq_Oct4_Ctrl_MWT_EarlyICM_9


title: RNA-seqOct4_Ctrl_MWT_LateICM_14


title: RNA-seq_Oct4_Ctrl_EarlyICM_6


title: RNA-seq_Oct4_Ctrl_EarlyICM_9


title: RNA-seq_Oct4_Ctrl_MWT_EarlyICM_7


title: RNA-seq_Oct4_Ctrl_MWT_EarlyICM_3


title: RNA-seq_Oct4_KO_Morula_12


title: RNA-seq_Oct4_Ctrl_MWT_EarlyICM_1


title: RNA-seq_Oct4_KO_Morula_8


title: RNA-seq_Oct4_Ctrl_MWT_EarlyICM_8


title: RNA-seq_Oct4_KO_Morula_4


title: RNA-seqOct4_Ctrl_MWT_LateICM_4


title: RNA-seq_Oct4_KO_EarlyICM_11


title: RNA-seq_Oct4_KO_Morula_5


title: RNA-seq_Oct4_KO_Morula_1


title: RNA-seq_Oct4_KO_Morula_7


title: RNA-seq_Oct4_KO_EarlyICM_5


title: RNA-seq_Oct4_KO_Morula_6


title: RNA-seq_Oct4_KO_EarlyICM_14


title: RNA-seq_Oct4_Ctrl_Morula_19


title: RNA-seq_Oct4_KO_Morula_2


title: RNA-seq_Oct4_Ctrl_Morula_18


title: RNA-seq_Oct4_Ctrl_Morula_1


title: RNA-seq_Oct4_Ctrl_Morula_7


title: RNA-seq_Oct4_Ctrl_Morula_4


title: RNA-seq_Oct4_Ctrl_MWT_Morula_6


title: RNA-seq_Oct4_Ctrl_MWT_Morula_10


title: RNA-seq_Oct4_Ctrl_MWT_Morula_8


title: RNA-seq_Oct4_Ctrl_MWT_Morula_5


title: RNA-seq_Oct4_Ctrl_Morula_11


title: RNA-seq_Oct4_Ctrl_MWT_Morula_1


title: ATAC-seq_Sox2_KO_LateICM_3

description:
Sox2_KO_LateICM_3
Accessible chromatin regions

data_processing:
Sequence reads were trimmed for adapter sequences using SeqPurge and trimmed reads no shorter than 20 bases were aligned to the mm10 mouse reference genome using Bowtie2 with a maximum fragment size of 2000. Duplicated reads were removed using Picard MarkDuplicates (https://broadinstitute.github.io/picard/) and only reads uniquely mapped to the standard chromosomes except chrY and chrM (mitochondrial genome) with mapping quality of at least 30 were used for the following analysis. Reads on the positive and negative strands were shifted +4 bp and −5 bp, respectively. Peak calling was performed using MACS2 with the following parameters: --keep-dup all -g mm --nomodel --shift -50 --extsize 100 -B --SPMR. Peaks that overlap with the blacklisted regions (https://sites.google.com/site/anshulkundaje/projects/blacklists; https://sites.google.com/site/atacseqpublic/atac-seq-analysis-methods/mitochondrialblacklists-1) and satellite repeats (RepeatMasker) were removed. Significant peak summits (q-value ≤0.001) from biological replicates were merged for each sample group using BEDTools with a maximum distance between two summits to be merged of 200 bp. Only merged peak summits that overlap with summits from at least two replicates were retained. Reads per million (RPM)-normalized pileup signals in the bedGraph format were converted into bigWig files using the UCSC bedGraphToBigWig tool. The bigWig files for the average signal of biological replicates were generated using the UCSC bigWigMerge tool. The heatmaps of RPM-normalized pileup signals around the TSSs of GENCODE (vM23) protein-coding genes were generated using deepTools.
For count-based analysis of transposon insertion events, merged peak regions were generated by combining the adjacent peak summits of all embryo groups and selecting 200-bp regions around the centers of each merged summit. 5’ ends of shifted read alignment on both the positive and negative strands were considered as transposon insertion sites. The number of transposon insertions in each merged peak region were counted for each sample using BEDTools and normalized and transformed to log2 scale using the rlog function of the Bioconductor package DESeq2 and significantly differential peaks were identified with an adjusted p-value cutoff of 0.05. Peaks located within 100 bp from TSSs of GENCODE (vM23) genes were considered as TSS-proximal peaks. The presence of TF binding motifs in merged peaks was investigated using FIMO with the default cutoff (p-value <10-4).
Assembly: mm10
Supplementary files format and content: bigwig
Supplementary files format and content: rlog of read count

extract_protocol:
All the embryos for ATAC-seq were collected from female mice superovulated with PMSG and hCG. Early and late morula samples were collected at E2.75 and E3.25, respectively. Early and late ICM samples were collected at E2.5 and cultured in KSOM with MEKi (PD0325901, 1 μM) until early and late blastocyst stages, respectively. ICM were isolated by immunosurgery.
20 to 40 morulae or ICMs were pooled and lysed in the lysis buffer (10 mM Tris·HCl, pH=7.4; 10 mM NaCl; 3 mM MgCl2; 0.15% Igepal CA-630; 0.1% Tween-20) for 10 min on ice. We added 0.1% Tween-20 to the lysis buffer as it greatly reduced reads mapped to mitochondrial DNA. Lyzed embryos were briefly washed, collected in 2.0 μl PBS containing 0.1 mg/ml polyvinyl alcohol (PVA) and transferred to 3.0 μl Tn5 transposome mixture (0.25 μl Tn5 transposome, 2.5 μl tagmentation buffer, Illumina, FC-121-1030; 0.25 μl H2O). The samples were incubated in 37 °C water bath for 30 min. Tagmentation was stopped by adding 2.0 μl 175 mM EDTA and incubated at 50 °C for 30 min. Excess EDTA was quenched by 2.0 μl 160 mM MgCl2. The libraries were amplified in the following reaction: 9.0 μl Transposed DNA, 10.0 μl NEBNext High-Fidelity 2×PCR Master Mix (New England Biolabs, M0541S), 0.25 μl 100 μM PCR Index 1, 0.25 μl 100 μM PCR Index 2 and 0.5 μl H2O. The sequences of index 1 and 2 are in Table EV2. The PCR program is as the following: 72 °C for 5 min; 98 °C for 30s; 16 thermocycles at 98 °C for 10 s, 63 °C for 30 s and 72 °C for 1 min; followed by 72 °C 5 min. Amplified libraries were purified twice with 1.2 × AMPure XP beads. The sequencing was performed on the NextSeq 500 system with pair-end 75bp.


title: ATAC-seq_Sox2_Ctrl_LateICM_4

description:
Sox2_Ctrl_LateICM_4
Accessible chromatin regions


title: ATAC-seq_Sox2_Ctrl_LateICM_2

description:
Sox2_Ctrl_LateICM_2
Accessible chromatin regions


title: ATAC-seq_Sox2_KO_LateICM_2

description:
Sox2_KO_LateICM_2
Accessible chromatin regions


title: RNA-seq_Oct4_Ctrl_MWT_Morula_3


title: ATAC-seq_Sox2_KO_LateICM_1

description:
Sox2_KO_LateICM_1
Accessible chromatin regions


title: ATAC-seq_Sox2_KO_EarlyICM_4

description:
Sox2_KO_EarlyICM_4
Accessible chromatin regions


title: ATAC-seq_Sox2_Ctrl_LateICM_1

description:
Sox2_Ctrl_LateICM_1
Accessible chromatin regions


title: ATAC-seq_Sox2_Ctrl_EarlyICM_4

description:
Sox2_Ctrl_EarlyICM_4
Accessible chromatin regions


title: ATAC-seq_Sox2_KO_EarlyICM_2

description:
Sox2_KO_EarlyICM_2
Accessible chromatin regions


title: ATAC-seq_Sox2_KO_EarlyICM_1

description:
Sox2_KO_EarlyICM_1
Accessible chromatin regions


title: RNA-seq_Oct4_Ctrl_Morula_16


title: ATAC-seq_Sox2_Ctrl_EarlyICM_2

description:
Sox2_Ctrl_EarlyICM_2
Accessible chromatin regions


title: RNA-seq_Oct4_Ctrl_MWT_Morula_7


title: ATAC-seq_Oct4_KO_LateICM_4

description:
Oct4_KO_LateICM_4
Accessible chromatin regions


title: ATAC-seq_Sox2_Ctrl_EarlyICM_3

description:
Sox2_Ctrl_EarlyICM_3
Accessible chromatin regions


title: RNA-seq_Oct4_Ctrl_Morula_15


title: ATAC-seq_Sox2_Ctrl_EarlyICM_1

description:
Sox2_Ctrl_EarlyICM_1
Accessible chromatin regions


title: ATAC-seq_Oct4_Ctrl_LateICM_4

description:
Oct4_Ctrl_LateICM_4
Accessible chromatin regions


title: ATAC-seq_Oct4_Ctrl_LateICM_2

description:
Oct4_Ctrl_LateICM_2
Accessible chromatin regions


title: ATAC-seq_Sox2_KO_LateICM_4

description:
Sox2_KO_LateICM_4
Accessible chromatin regions


title: ATAC-seq_Oct4_KO_EarlyICM_4

description:
Oct4_KO_EarlyICM_4
Accessible chromatin regions


title: ATAC-seq_Sox2_KO_EarlyICM_3

description:
Sox2_KO_EarlyICM_3
Accessible chromatin regions


title: ATAC-seq_Sox2_Ctrl_LateICM_3

description:
Sox2_Ctrl_LateICM_3
Accessible chromatin regions


title: ATAC-seq_Oct4_KO_LateICM_2

description:
Oct4_KO_LateICM_2
Accessible chromatin regions


title: ATAC-seq_Oct4_Ctrl_LateMorula_3

description:
Oct4_Ctrl_LateMorula_3
Accessible chromatin regions

source_name: E3.25 late morula

value: E3.25 late morula


title: ATAC-seq_Oct4_Ctrl_LateICM_3

description:
Oct4_Ctrl_LateICM_3
Accessible chromatin regions


title: ATAC-seq_Oct4_Ctrl_EarlyICM_4

description:
Oct4_Ctrl_EarlyICM_4
Accessible chromatin regions


title: ATAC-seq_Oct4_Ctrl_LateICM_1

description:
Oct4_Ctrl_LateICM_1
Accessible chromatin regions


title: ATAC-seq_Oct4_Ctrl_EarlyICM_3

description:
Oct4_Ctrl_EarlyICM_3
Accessible chromatin regions


title: ATAC-seq_Oct4_Ctrl_LateMorula_1

description:
Oct4_Ctrl_LateMorula_1
Accessible chromatin regions


title: ATAC-seq_Oct4_Ctrl_EarlyICM_2

description:
Oct4_Ctrl_EarlyICM_2
Accessible chromatin regions


title: ATAC-seq_Oct4_KO_EarlyICM_2

description:
Oct4_KO_EarlyICM_2
Accessible chromatin regions


title: ATAC-seq_Oct4_KO_EarlyICM_1

description:
Oct4_KO_EarlyICM_1
Accessible chromatin regions


title: ATAC-seq_Oct4_Ctrl_EarlyICM_1

description:
Oct4_Ctrl_EarlyICM_1
Accessible chromatin regions


title: ATAC-seq_Oct4_KO_Morula_3

description:
Oct4_KO_Morula_3
Accessible chromatin regions


title: ATAC-seq_Oct4_KO_LateMorula_3

description:
Oct4_KO_LateMorula_3
Accessible chromatin regions


title: ATAC-seq_Oct4_Ctrl_Morula_2

description:
Oct4_Ctrl_Morula_2
Accessible chromatin regions


title: ATAC-seq_Oct4_KO_LateICM_1

description:
Oct4_KO_LateICM_1
Accessible chromatin regions


title: ATAC-seq_Oct4_KO_LateMorula_1

description:
Oct4_KO_LateMorula_1
Accessible chromatin regions


title: ATAC-seq_Oct4_Ctrl_Morula_3

description:
Oct4_Ctrl_Morula_3
Accessible chromatin regions


title: ATAC-seq_Oct4_Ctrl_LateMorula_2

description:
Oct4_Ctrl_LateMorula_2
Accessible chromatin regions


title: ATAC-seq_Oct4_Ctrl_Morula_1

description:
Oct4_Ctrl_Morula_1
Accessible chromatin regions


title: RENCA anti-PD-L1 Resistant met rep 2

city: Buffalo

first: John

last: Ebos

state: NY

address: Elm & Carlton Streets

department: Department of Cancer Genetics and Genomics

institute: Roswell Park Cancer Institute

data_processing:
Pre-alignment quality control was performed using FASTQC v0.11.5.
Sequenced reads (fastq files) were alinged to the mouse genome GRCm38 M16 (Gencode) using STAR v2.6.0a.
Post-alignment quality control was performed using RSeQC v2.6.5.
Estimated counts at the gene level were quantified using RSEM v1.3.1.
Genome_build: GRCm38
Supplementary_files_format_and_content: tab-delimited text file include RSEM estimated counts at the gene level for all samples

source_name: Kidney cancer cells

growth_protocol: Cells were maintained in RPMI supplemented with 5% FBS with 0.5μg/ml of isotype control or anti-PD-L1. Growth media was refreshed every 3-4 days.

extract_protocol:
Total RNA from individual samples was extracted using the RNeasy mini kit (QIAGEN; 74104). Genomic DNA was digested using DNase I (QIAGEN; 79254) as described in the on-column DNase digestion protocol from QIAGEN.
One microgram total RNA was used for library preparation using the TruSeq Stranded mRNA kit (Illumina Inc) following manufacturer's instructions.

tag: variant

value: RENCA PDR

tag: dose

tag: treatment duration

value: >4 weeks


title: RENCA anti-PD-L1 Resistant met rep 1


title: RENCA anti-PD-L1 Resistant met rep 3


title: ATAC-seq_Oct4_KO_Morula_2

description:
Oct4_KO_Morula_2
Accessible chromatin regions


title: ATAC-seq_Oct4_KO_EarlyICM_3

description:
Oct4_KO_EarlyICM_3
Accessible chromatin regions


title: ATAC-seq_Oct4_KO_Morula_1

description:
Oct4_KO_Morula_1
Accessible chromatin regions


title: ATAC-seq_Oct4_KO_LateMorula_2

description:
Oct4_KO_LateMorula_2
Accessible chromatin regions


title: RENCA anti-PD-L1 Resistant rep 2


title: RENCA Parental met rep 2

value: RENCA

value: Isotype Control


title: RENCA anti-PD-L1 Resistant rep 3


title: RENCA Parental met rep 3


title: RENCA anti-PD-L1 Resistant rep 1


title: RENCA Parental rep 3


title: RENCA Parental rep 1


title: EMT6 anti-PD-L1 resistant rep 2

source_name: Breast tumor tissue

growth_protocol: EMT6 mouse breast tumors implanted orthotopically in Balb/c mice

value: EMT6 PDR

value: Until institutional endpoint


title: EMT6 vitro Parental rep 2

source_name: Breast cancer cells


title: EMT6 vitro anti-PD-L1 Resistant rep 2


title: EMT6 vitro anti-PD-L1 Resistant rep 3


title: EMT6 vitro anti-PD-L1 Resistant rep 1


title: EMT6 Control rep 3


title: EMT6 Control rep 2


title: EMT6 vitro Parental rep 1


title: EMT6 vitro Parental rep 3


title: EMT6 anti-PD-L1 resistant met


title: EMT6 anti-PD-L1 resistant rep 3


title: H3.3K36RH3.2HWT, Rep2

city: Chapel Hill

first: Greg

last: Matera

state: NC

address: 3352 Genome Sciences Building

institute: UNC Chapel Hill

data_processing:
Reads were quality trimmed using bbmap v38 bbduk qtrim=rl trimq=20
Reads were aligned using STAR v.2.7.2a to DM6.38
Raw count table was generated using subread v.2.0.3 using DM6.38 with the following parameters: -g gene_id -p -s 1
Assembly: DM6
Supplementary files format and content: Tab-delimited text file includes raw counts for each sample.

organism: Drosophila melanogaster

source_name: Whole L1, mixed sex

growth_protocol: Embryos from crosses kept at 25 degrees C, were laid on grape-juice agar plates and harvested for RNA collection at the L1 stage.

extract_protocol:
25-30 larvae were collected in 1ml Trizol. RNA was isolated from the aqueous phase using the Zymo Clean and Concentrator Kit (with DNASEI digestion).
Total RNA Seq libraries were prepared with Nugen Ovation Universal Drosophila kit

value: Whole L1

tag: Sex

value: pooled male and female


title: H3.3K36RH3.2HWT, Rep1

tag: treatment


title: EMT6 anti-PD-L1 resistant rep 1


title: H3.3K36RH3.2K36R, Rep2


title: H3.3K36RH3.2HWT, Rep3


title: EMT6 Control rep 1