title: HeLa_dsRNA_shTRMT1-rep2

description:
cell culture
Sample 36
Misincorporation_in-vivo_dsRNA_AllMethyl.xlsx

data_processing:
Library strategy: DAMM-seq
The sequencing data was all trimmed with cutadapt tool to remove adapters and low-quality reads (length shorter than 18). Reads were processed with BBMap tool (https://sourceforge.net/projects/bbmap/) to remove PCR duplicates, and then further trimmed the random barcode using cutadapt tool and remove low-quality reads again. Reads were then aligned to the relevant genome (human-hg19) using Tophat2 (version 2.1.1) and bowtie2 (version 2.3.5.1) with at most three mismatches. The generated .bam files were split into positive strand and negative strand using Samtools, and then sorted. Identification of sequence variants was conducted by measuring the base composition at each position using fine-tuned bam-readcount (https://github.com/genome/bam-readcount). The generated bam-readcount results were parsed and analyzed by in-house scripts. Misincorporation ratio at each methylated site (m1A, m1G, m22G, m3C) in dsRNA were calculated by data output from bam-readcount pipeline, and also confirmed by direct visualization through IGV software.
Genome_build: hg19
Supplementary_files_format_and_content: Misincorporation_in-vivo_dsRNA_AllMethyl.xlsx

growth_protocol: Human HepG2 and HeLa cell lines used in this study were all purchased from ATCC (the American Type Culture Collection). HeLa cell line was grown in DMEM (GIBCO,11965) media supplemented with 10% FBS and 1% 100X Pen/Strep (GIBCO), while HepG2 cell line was maintained in DMEM (GIBCO, 11995), supplemented with 10% FBS and 1% 100X Pen/Strep (GIBCO). All cells were cultured at 37°C under 5.0% CO2, at Heracell™ VIOS 160i incubator (Thermo Scientific).

extract_protocol:
Twenty 15-cm plates of HepG2 cells were prepared for 48-hour knockdown (siControl, siALKBH7, 5 plates per replicate) or twenty 15-cm plates of HeLa cells were prepared for 24-hour overexpression (o/e Control, o/e ALKBH7, 5 plates per replicate) or twenty 15-cm plates of HepG2 cells were prepared for wild-type vs. ALKBH7 knockout (WT, ALKBH7 KO, 5 plates per replicate). At first, for every 5 plates of cells, 250 uL Protein G Dynabeads were washed twice and then resuspended in 1 mL IP wash buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM MgCl2, 0.5% NP-40). 25 µg of anti-dsRNA mAb (J2) was incubated with such 250 µL of beads for 1 hour at room temperature and then moved to 4 °C for another 1-2 hours. During this incubation, for every 5 plates, the cell culture media was removed, and cells were washed by cold DPBS. Then cells were collected by cell lifter in 5 mL cold DPBS and merged into one 50 mL centrifuge tube. The cell suspension was spun at 300g at 4 °C for 2 min and the cell pellet was resuspended with 1 mL NP-40 lysis buffer prepared in 10 mL volume: 50 mM HEPES pH 7.5, 150 mM NaCl, 2 mM EDTA, 10 mM MgCl2, 0.5% NP-40, plus 50 uL TURBO™ DNase (2 U/µL, AM2238, Invitrogen) and 500 uL SUPERase•In™ RNase Inhibitor (20 U/μL, AM2694, Invitrogen). Incubate the cell suspension at 4 °C for 30 minutes, followed by a spin at 16,000g at 4 °C for 5 min. The supernatant was transferred to a new Eppendorf tube and keep 10 uL of that as input with TRIzol added. The 1 mL lysate was dilute to 5 mL with IP wash buffer and then 250 uL of J2-Dynabeads was added for an incubation at 4 °C for 2 hours. The magnetic beads were washed once with 1 ml of IP wash buffer and transferred into a new Eppendorf tube by 500 µL of IP wash buffer. The beads were further washed twice by 1 mL of IP wash buffer, and J2-bound dsRNA was extracted with TRIzol reagent.
Around 50 ng human cellular double-stranded RNA by J2-IP (dsNRA) was fragmented into 40-50 nt using RNA Fragmentation Reagent (AM8740, Invitrogen) following the manufacturer’s protocol and Oligo Clean & Concentrator (Zymo Research). These RNAs can be from diverse cellular treatments such as siALKBH7 vs. siControl or o/e Control vs. o/e ALKBH7 or wild-type vs. ALKBH7 KO. Then the library construction procedures will be conducted with T4 PNK end repair, RNA 3’-adapter ligation, HIV RT reverse transcription, cDNA 3’-adapter ligation and PCR amplification respectively, according to standard steps in section “DAOMM-seq for methylated site detection”. All libraries were sequenced on Illumina NovaSeq 6000 with single-end 100 bp read length.

treatment_protocol: siRNA knockdown and plasmid transfection. Typical 15-cm plates of human HepG2 cells were prepared for 48-hour ALKBH7 knockdown. AllStars negative control siRNA from Qiagen (1027281) was used as control siRNA (siControl). Human ALKBH7 siRNA was ordered from Qiagen with catalog number as Hs_SPATA11_3 FlexiTube siRNA (SI00730793). Transfection was achieved by using Lipofectamine RNAiMAX (Invitrogen) for siRNA. The plasmid for human ALKBH7 overexpression was purchased from VectorBuilder with Vector ID: VB180629-1142wpx. Typical 15-cm plates of human HeLa cells were prepared for 24-hour ALKBH7 overexpression, where Lipofectamine LTX Plus (Invitrogen) was used for plasmid transfection following the manufacturer’s protocols. shRNA knockdown. The TRMT1(TRCN0000038765) and control vectors were purchased from Horizon Discovery. Plasmids pMD2.G and psPAX2 were purchased from Addgene. All the high-purify plasmids used for transduction were prepared with HiSpeed Plasmid Maxi Kit (Qiagen). HEK293T cells were cultured with DMEM 11965 with 10% v/v FBS and 1% Pen Strep. When 10-cm 293T cells were under 80% confluency, the lentiviral vectors and the packaging vectors were prepared in an equimolar ratio with 10 µg in total mixed with 300 µL Opti-MEM. And 30 µL PEI-MAX (Polysciences, dissolved to 1mg/mL with water and pH adjusted to 7) were suspended in 300 µL Opti-MEM. Both were incubated for 5-10 min at room temperature and then mixed for another 5-10 min incubation. The medium of 293T were aspirated and replaced with 4 mL Opti-MEM before the lentiviral vector mix was added. After 4-6 hours, the medium was changed to culture medium. After the two-day culture of the cells, the supernatant medium was collected and centrifuged at 2000 rpm for 3 min to remove the cell debris as the pellet. Collect the clear supernatant and the virus were the precipitated with PEG-it Virus Precipitation Solution (System Biosciences). After the overnight incubation at 4 ℃, the lentivirus solution was centrifuged at 1500 g for 30 min and the supernatant was discharged without disturbing the viral pellet. The pellet could be stored at -80 ℃ for further use. The pellets were suspended with culture medium and the TransDux MAX Lentivirus Transduction Enhancer (System Biosciences) according to the manufactory’s protocol and then added to the HeLa cells in 6-well plate at 70% confluency. The cells were cultured and selected with 1 µg/mL puromycin.

value: HeLa


title: HeLa_dsRNA_overexpression_Control-rep1

status: Public on Jun 02 2021

type: SRA

city: Chicago

first: Li-Sheng

last: Zhang

state: IL

address: 929 E 57th St Rm E313

department: Department of Chemistry

country: USA

institute: The University of Chicago

description:
cell culture
Sample 37
Misincorporation_in-vivo_dsRNA_AllMethyl.xlsx

library_source: transcriptomic

molecule: total RNA

organism: Homo sapiens

tag: cell line

tag: genotype

tag: rna fraction

value: mitochondrial dsRNA


title: HeLa_dsRNA_shTRMT1-rep1

description:
cell culture
Sample 35
Misincorporation_in-vivo_dsRNA_AllMethyl.xlsx


title: HeLa_dsRNA_overexpression_Control-rep2

description:
cell culture
Sample 38
Misincorporation_in-vivo_dsRNA_AllMethyl.xlsx


title: HeLa_dsRNA_shControl-rep2

description:
cell culture
Sample 34
Misincorporation_in-vivo_dsRNA_AllMethyl.xlsx

value: shControl


title: 23_1

status: Public on Jun 11 2021

city: New York

first: Sander

middle: M

last: Houten

state: NY

address: 1425 Madison Avenue, Box 1498

department: Genetics and Genomic Sciences

institute: Icahn School of Medicine at Mount Sinai

description: RNA isolation batch 1

data_processing:
Illumina HiSeq2500 for base caling
Read alignment. STAR (release 2.5.1b)
gene-level read quantification via featureCounts v1.5.3
Genome_build: mouse genome mm10 (GRCm38.75)
Supplementary_files_format_and_content: text file containing raw gene counts for each sample

molecule: polyA RNA

organism: Mus musculus

source_name: heart

extract_protocol:
RNA was isolated using QIAzol lysis reagent followed by purification using the RNeasy kit (Qiagen)
mRNA-focused cDNA libraries were generated using Illumina reagents (polyA capture), and samples were run on an Illumina HiSeq 2500 sequencer to yield a read depth of approximately 70 million 100 nucleotide single-end reads per samples.

tag: strain

value: mixed (mostly C57BL/6N with some 129)

tag: tissue

value: heart

tag: Sex

value: male

value: Acadl KO / Acadvl Het

tag: diet

value: chow


title: HepG2_dsRNA_ALKBH7_KO-rep1

description:
cell culture
Sample 31
Misincorporation_in-vivo_dsRNA_AllMethyl.xlsx


title: HepG2_dsRNA_siALKBH7-rep2

description:
cell culture
Sample 28
Misincorporation_in-vivo_dsRNA_AllMethyl.xlsx


title: HepG2_dsRNA_ALKBH7_WT-rep2

description:
cell culture
Sample 30
Misincorporation_in-vivo_dsRNA_AllMethyl.xlsx


title: HepG2_dsRNA_ALKBH7_KO-rep2

description:
cell culture
Sample 32
Misincorporation_in-vivo_dsRNA_AllMethyl.xlsx


title: icSHAPE_siControl_Pulldown-rep1

description:
cell culture
Sample 45
icSHAPE_ReadsCoverage_mt-RNA_H-strand.xlsx,  icSHAPE_ReadsCoverage_mt-RNA_L-strand.xlsx

data_processing:
Library strategy: icSHAPE
The sequencing data was all trimmed with cutadapt tool to remove adapters and low-quality reads (length shorter than 18). Reads were processed with BBMap tool (https://sourceforge.net/projects/bbmap/) to remove PCR duplicates, and then further trimmed the random barcode using cutadapt tool and remove low-quality reads again. Reads were then aligned to the relevant genome (human-hg19) using Tophat2 (version 2.1.1) and bowtie2 (version 2.3.5.1) with at most three mismatches. The generated .bam files were split into positive strand and negative strand using Samtools, and then sorted. The total reads coverage at each position was calculated by fine-tuned bam-readcount (https://github.com/genome/bam-readcount). The generated bam-readcount results were used for calculating enrichment folds of the flexible region icSHAPE peak.
Genome_build: hg19
Supplementary_files_format_and_content: icSHAPE_ReadsCoverage_mt-RNA_H-strand.xlsx,  icSHAPE_ReadsCoverage_mt-RNA_L-strand.xlsx

growth_protocol: Human HepG2 cell line used in this study were all purchased from ATCC (the American Type Culture Collection). HepG2 cell line was maintained in DMEM (GIBCO, 11995), supplemented with 10% FBS and 1% 100X Pen/Strep (GIBCO). All cells were cultured at 37°C under 5.0% CO2, at Heracell™ VIOS 160i incubator (Thermo Scientific).

extract_protocol:
. Forty 15-cm plates of HepG2 cells were prepared for 48-hour knockdown (siControl, siALKBH7, 5 plates per replicate). Upon the completion of knockdown, cells were collected by cell lifter in 5 mL cold DPBS and merged into one 50 mL centrifuge tube. The cell suspension was spun at 300g at 4 °C for 2 min to produce the cell pellet. For the cell pellet corresponding to one of four replicates in each case (4 of siControl or 4 of siALKBH7), add 500 uL control solution (450 uL DPBS + 50 uL DMSO) into two replicates of each case respectively, and add 500 uL NAI-N3 solution (450 uL DPBS + 50 uL 1.0 M NAI-N3 in DMSO) into other two replicates of each case respectively. Then incubate the resuspended solution at 37 ℃ for 5 min, followed by centrifuge at 300g for 2 min. Remove the supernatant carefully and wash the cell pellet with cold DPBS once, followed by centrifuge at 300g for 2 min again.   The cell pellet was resuspended with 1 mL NP-40 lysis buffer prepared in 10 mL volume: 50 mM HEPES pH 7.5, 150 mM NaCl, 2 mM EDTA, 10 mM MgCl2, 0.5% NP-40, plus 50 uL TURBO™ DNase (2 U/µL, AM2238, Invitrogen) and 500 uL SUPERase•In™ RNase Inhibitor (20 U/μL, AM2694, Invitrogen). Incubate the cell suspension at 4 °C for 30 minutes, followed by a spin at 16,000g at 4 °C for 5 min. The supernatant was transferred to a new Eppendorf tube and keep 10 uL of that as input with TRIzol added. The 1 mL lysate was dilute to 5 mL with IP wash buffer and then 250 uL of J2-Dynabeads was added for an incubation at 4 °C for 2 hours. The magnetic beads were washed once with 1 ml of IP wash buffer and transferred into a new Eppendorf tube by 500 µL of IP wash buffer. The beads were further washed twice by 1 mL of IP wash buffer, and J2-bound dsRNA was extracted with TRIzol reagent.
Dilute the harvested dsRNA in 17 uL RNase-free water (for both DMSO-treated and NAI-N3 treated samples), and then add 2 uL of 2 mM sDIBO-biotin together with 1 uL SUPERase•In™ RNase Inhibitor. Incubate at 37 °C for 1 hour and purify RNA through Oligo Clean & Concentrator (Zymo Research). Dilute the purified RNA with 17 uL RNase-free water and add 2 uL RNA Fragmentaion Buffer (NEBNext® Magnesium RNA Fragmentation Module), followed by heating at 95 °C for 3 min. Put all samples onto ice immediately and add 2 uL Stop Buffer. RNA purification was performed for the 4 samples from DMSO treated case using Oligo Clean & Concentrator (Zymo Research), to afford “input” samples. For other 4 samples from NAI-N3 treated case, mix the RNA solution with 20 uL freshly-prepared C1 beads suspension (10 uL Dynabeads™ MyOne™ Streptavidin C1 beads washed twice with 200 uL 1X B&W buffer and stored in 20 uL 2X B&W buffer based on the manufacturer’s protocol). After the incubation of 30 min at 4 °C, wash the beads three times with icSHAPE wash buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 0.05% NP40), followed by proteinase K digestion at 55 °C for 30 min. The harvested RNA (serving as “pulldown”, from NAI-N3 treated) was purified by Oligo Clean & Concentrator (Zymo Research), and sent for library construction together with “input” samples using SMARTer Stranded Total RNA-Seq Kit v2 (Takara). All libraries were sequenced on Illumina Illumina NovaSeq 6000 with single-end 100 bp read length.

treatment_protocol: siRNA knockdown and plasmid transfection. Typical 15-cm plates of human HepG2 cells were prepared for 48-hour ALKBH7 knockdown. AllStars negative control siRNA from Qiagen (1027281) was used as control siRNA (siControl). Human ALKBH7 siRNA was ordered from Qiagen with catalog number as Hs_SPATA11_3 FlexiTube siRNA (SI00730793). Transfection was achieved by using Lipofectamine RNAiMAX (Invitrogen) for siRNA.

value: siControl

tag: treatment

value: Pulldown


title: HepG2_dsRNA_siALKBH7-rep1

description:
cell culture
Sample 27
Misincorporation_in-vivo_dsRNA_AllMethyl.xlsx


title: HeLa_dsRNA_overexpression_ALKBH7-rep2

description:
cell culture
Sample 40
Misincorporation_in-vivo_dsRNA_AllMethyl.xlsx


title: HeLa_tRNA_overexpression_ALKBH7-rep2

description:
cell culture
Sample 24
Misincorporation_in-vivo_mt-tRNA_AllMethyl.xlsx

data_processing:
Library strategy: DAMM-seq
The sequencing data was all trimmed with cutadapt tool to remove adapters and low-quality reads (length shorter than 18). Reads were processed with BBMap tool (https://sourceforge.net/projects/bbmap/) to remove PCR duplicates, and then further trimmed the random barcode using cutadapt tool and remove low-quality reads again. Reads were then aligned to the relevant genome (human-hg19) using Tophat2 (version 2.1.1) and bowtie2 (version 2.3.5.1) with at most three mismatches. The generated .bam files were split into positive strand and negative strand using Samtools, and then sorted. Identification of sequence variants was conducted by measuring the base composition at each position using fine-tuned bam-readcount (https://github.com/genome/bam-readcount). The generated bam-readcount results were parsed and analyzed by in-house scripts. Misincorporation ratio at each methylated site (m1A, m1G, m22G, m3C) in tRNA were calculated by data output from bam-readcount pipeline, and also confirmed by direct visualization through IGV software.
Genome_build: hg19
Supplementary_files_format_and_content: Misincorporation_in-vivo_mt-tRNA_AllMethyl.xlsx

extract_protocol:
Basically, as the first step of harvesting cells, the cells were washed once with DPBS buffer for each plate after aspirating the media. Then cellular total RNA was isolated with TRIzol reagent (Invitrogen) following manufacturer’s protocol by isopropanol precipitation. The small RNA fraction (size less than 200 nt) was further extracted from the prepared cellular total RNA by mirVana™ miRNA Isolation Kit (AM1560, Invitrogen) protocols.
Around 50 ng human cellular small RNA (size less than 200 nt) was fragmented into 40-50 nt using RNA Fragmentation Reagent (AM8740, Invitrogen) following the manufacturer’s protocol and Oligo Clean & Concentrator (Zymo Research). These RNAs can be from diverse cellular treatments such as siALKBH7 vs. siControl or o/e Control vs. o/e ALKBH7. Then the library construction procedures will be conducted with T4 PNK end repair, RNA 3’-adapter ligation, HIV RT reverse transcription, cDNA 3’-adapter ligation and PCR amplification respectively, according to standard steps in section “DAOMM-seq for methylated site detection”. All libraries were sequenced on Illumina NovaSeq 6000 with single-end 100 bp read length.

value: mitochondrial tRNA


title: HeLa_dsRNA_overexpression_ALKBH7-rep1

description:
cell culture
Sample 39
Misincorporation_in-vivo_dsRNA_AllMethyl.xlsx


title: HepG2_dsRNA_siControl-rep1

description:
cell culture
Sample 25
Misincorporation_in-vivo_dsRNA_AllMethyl.xlsx


title: HeLa_tRNA_overexpression_ALKBH7-rep1

description:
cell culture
Sample 23
Misincorporation_in-vivo_mt-tRNA_AllMethyl.xlsx


title: HeLa_tRNA_overexpression_Control-rep1

description:
cell culture
Sample 21
Misincorporation_in-vivo_mt-tRNA_AllMethyl.xlsx


title: icSHAPE_siControl_Input-rep2

description:
cell culture
Sample 42
icSHAPE_ReadsCoverage_mt-RNA_H-strand.xlsx,  icSHAPE_ReadsCoverage_mt-RNA_L-strand.xlsx

value: Input


title: HeLa_tRNA_overexpression_Control-rep2

description:
cell culture
Sample 22
Misincorporation_in-vivo_mt-tRNA_AllMethyl.xlsx


title: HeLa_tRNA_shControl-rep2

description:
cell culture
Sample 18
Misincorporation_in-vivo_mt-tRNA_AllMethyl.xlsx


title: HeLa_tRNA_shTRMT1-rep2

description:
cell culture
Sample 20
Misincorporation_in-vivo_mt-tRNA_AllMethyl.xlsx


title: HeLa_tRNA_shControl-rep1

description:
cell culture
Sample 17
Misincorporation_in-vivo_mt-tRNA_AllMethyl.xlsx


title: HepG2_dsRNA_siControl-rep2

description:
cell culture
Sample 26
Misincorporation_in-vivo_dsRNA_AllMethyl.xlsx


title: HeLa_tRNA_shTRMT1-rep1

description:
cell culture
Sample 19
Misincorporation_in-vivo_mt-tRNA_AllMethyl.xlsx


title: HepG2_tRNA_siALKBH7-rep1

description:
cell culture
Sample 15
Misincorporation_in-vivo_mt-tRNA_AllMethyl.xlsx


title: HepG2_tRNA_siControl-rep2

description:
cell culture
Sample 14
Misincorporation_in-vivo_mt-tRNA_AllMethyl.xlsx


title: HepG2_tRNA_siALKBH7-rep2

description:
cell culture
Sample 16
Misincorporation_in-vivo_mt-tRNA_AllMethyl.xlsx


title: HeLa_dsRNA_shControl-rep1

description:
cell culture
Sample 33
Misincorporation_in-vivo_dsRNA_AllMethyl.xlsx


title: HepG2_dsRNA_ALKBH7_WT-rep1

description:
cell culture
Sample 29
Misincorporation_in-vivo_dsRNA_AllMethyl.xlsx


title: E56_3b

status: Public on Mar 26 2021

data_processing:
Illumina HiSeq2500 for base caling
Read alignment. STAR
gene-level read quantification via featureCounts
Filtering raw reads: 1 cpm in at least 2 samples
Normalization via calcNormFactors of library edgeR
Genome_build: mouse genome mm10 (GRCm38.75)
Supplementary_files_format_and_content: text file containing filtered, normalized counts per million reads according to normalization factors for Ehhadh WT and Ehhadh KO samples
Supplementary_files_format_and_content: text file containing raw gene counts for each sample including 129S2 and DBA/2J

source_name: kidney

extract_protocol:
RNA was isolated using QIAzol lysis reagent followed by purification using the RNeasy kit (Qiagen)
mRNA-focused cDNA libraries were generated using Illumina reagents (polyA capture), and samples were run on an Illumina HiSeq 2500 sequencer to yield a read depth of approximately 56 million 100 nucleotide single-end reads per samples.

value: kidney

tag: ehhadh genotype

value: ko


title: HepG2_tRNA_siControl-rep1

description:
cell culture
Sample 13
Misincorporation_in-vivo_mt-tRNA_AllMethyl.xlsx


title: HepG2_SmallRNA_Demethylated-rep2

description:
cell culture
Sample 4
DAMM-seq_mt-tRNA_AllMethyl.xlsx

data_processing:
Library strategy: DAMM-seq
We termed this approach “Demethylation-Assisted Multiple Methylation sequencing” (DAMM-seq). The sequencing data was all trimmed with cutadapt tool to remove adapters and low-quality reads (length shorter than 18). Reads were processed with BBMap tool (https://sourceforge.net/projects/bbmap/) to remove PCR duplicates, and then further trimmed the random barcode using cutadapt tool and remove low-quality reads again. Reads were then aligned to the relevant genome (human-hg19) using Tophat2 (version 2.1.1) and bowtie2 (version 2.3.5.1) with at most three mismatches. The generated .bam files were split into positive strand and negative strand using Samtools, and then sorted. Identification of sequence variants was conducted by measuring the base composition at each position using fine-tuned bam-readcount (https://github.com/genome/bam-readcount). The generated bam-readcount results were parsed and analyzed by in-house scripts. Misincorporation ratio at each methylated site (m1A, m1G, m22G, m3C) in tRNA were calculated by data output from bam-readcount pipeline, and also confirmed by direct visualization through IGV software.
Genome_build: hg19
Supplementary_files_format_and_content: All DAMM-seq detected mitochondrial tRNA methylation sites are described in the processed file named DAMM-seq_mt-tRNA_AllMethyl.xlsx.

extract_protocol:
All RNA extracted from wild-type cells inclusing HepG2, HeLa and HEK293T cell lines. Basically, as the first step of harvesting cells, the cells were washed once with DPBS buffer for each plate after aspirating the media. Then cellular total RNA was isolated with TRIzol reagent (Invitrogen) following manufacturer’s protocol by isopropanol precipitation. The small RNA fraction (size less than 200 nt) was further extracted from the prepared cellular total RNA by mirVana™ miRNA Isolation Kit (AM1560, Invitrogen) protocols.
Around 300 ng human cellular small RNA (size less than 200 nt) was fragmented into 40-50 nt using RNA Fragmentation Reagent (AM8740, Invitrogen) following the manufacturer’s protocol and Oligo Clean & Concentrator (Zymo Research). Save ~50 ng as “input” and perform enhanced demethylation treatment towards the rest fragmented RNA with 1.0 uL AlkB mutant (10 mg/mL protein concentration), followed by purification using Oligo Clean & Concentrator (Zymo Research). Then the “input” RNA and demethylase treated RNA were sent for end repair mediated by T4 Polynucleotide Kinase (EK0032, Thermo Fisher Scientific): RNA was mixed with 5 uL 10X T4 Polynucleotide Kinase Reaction Buffer (B0201S, NEB) and 5 uL T4 PNK, diluted to a final volume of 50 uL and incubated at 37 °C for 1 hour followed by Oligo Clean & Concentrator (Zymo Research). Starting with 3’-adaptor ligation, repaired RNA fragments were incubated with 1.0 uL 50 uM 3’-linker (5’App-AGATCGGAAGAGCGTCGTG-3SpC3) at 70 °C for 2 min and transferred to ice immediately. 2,5 uL 10X T4 RNA Ligase Reaction Buffer (NEB), 7.5 uL 50% PEG8000, 1 uL SUPERase•In™ RNase Inhibitor and 2 uL T4 RNA ligase 2 truncated KQ (NEB) were added accordingly to the RNA-adapter mixture, then the reaction was diluted to a final volume of 25 uL and incubated at 25 °C for 2 hours followed by 16 °C for 12 hours. The completed ligation reaction was further diluted into 47 uL by Nuclease-free water, and the excessive adapters were removed with 2 uL 5’-Deadenylase (NEB) at 30 °C for 1 hour followed by adding 1.0 uL RecJf (NEB) for ssDNA digestion at 37 °C for 1 hour. The 3’-end ligated RNA was extracted by RNA Clean & Concentrator-5 (Zymo Research).
The purified RNA was incubated with 1.0 uL of 2.0 uM RT primer (5’-ACACGACGCTCTTCCGATCT-3’) at 65 °C for 2 min and moved onto ice immediately. Then 2 uL 10X AMV Reverse Transcriptase Reaction Buffer (NEB), 2 uL 10 mM dNTP Solution Mix (NEB), 1 uL RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific) and 2 uL HIV Recombinant Reverse Transcriptase (Worthington) were added into the RNA-primer mixture, followed by a one-hour incubation (diluted into 20 uL volume) at 37 °C for 90 min. The RT enzymes were deactivated at 70 °C for 5 min then kept on ice. The harvested cDNAs were purified and collected by Oligo Clean & Concentrator (Zymo Research). Then the mixture of cDNA and 1.0 uL of 50 uM cDNA 3’-linker (5’Phos-NNNNNNAGATCGGAAG AGCACACGTCTG-3SpC3) were denatured at 75 °C for 2 min and then put onto ice. 2.5 uL 10X T4 RNA Ligase Reaction Buffer (NEB), 12.5 uL 50% PEG8000, 2.5 uL 10 mM ATP and 1.0 uL T4 RNA ligase 1 (high concentration, NEB) were added accordingly to cDNA-adapter mixture, then the reaction was incubated at 25 °C for 12 hours.
Finally, the libraries were amplified using the universal and indexed primers from NEBNext Multiplex Oligos for Illumina (NEB). All libraries were sequenced on Illumina NovaSeq 6000 with single-end 100 bp read length.

value: AlkB-treated

value: cellular small RNA


title: HepG2_SmallRNA_Input-rep1

description:
cell culture
Sample 1
DAMM-seq_mt-tRNA_AllMethyl.xlsx

value: untreated


title: HepG2_SmallRNA_Input-rep2

description:
cell culture
Sample 2
DAMM-seq_mt-tRNA_AllMethyl.xlsx


title: E54_1b

value: wt


title: DBA_2J


title: E55_7b


title: 64_3


title: E54_5b


title: 64_2


title: 63_3


title: 129S2_SvPasCrl


title: 63_2


title: A64_DT_0.01ng of synthetic DNA in 10ng of AML sample: Patient 5

status: Public on Feb 16 2021

city: Toronto

first: Michael

last: Hoffman

state: ON

address: 101 College St.

department: Research

country: Canada

institute: Princess Margaret Cancer Centre

description:
AML plasma + synthetic spike-in DNA
Multiple identifiers for this sample are: 6658, A64, A64_DT, Sample 1, Princess Margaret Cancer Biobank ID 150197

library_source: genomic

data_processing:
Library strategy: cfMeDIP-seq
Adapters were trimmed using fastp v. 0.11.5
Alignment to our synthetic fragments and subsequently to hg38 was performed using BowTie2 v. 2.4.5.
Genome_build: hg38 pr alignment to synthetic fragment sequence
Supplementary_files_format_and_content: bed
The column headers for the bed files are:
1. read 1 chromosome or scaffold
2. read 1 start position
3. read 1 end position
4. read 2 chromosome or scaffold
5. read 2 start position
6. read 2 end position
7. strand
8. strand
9. read count

molecule: genomic DNA

source_name: AML plasma + synthetic spike-in DNA

growth_protocol: HCT116 cells were grown with McCoy Media supplemented with 10% FBS and 1% Penicillin/Streptomycin in 75cm2 flasks at 37˚C and 5% CO2

extract_protocol:
The genomic DNA of the collected cells were extracted using Thermofisher PureLink® Genomic DNA Kit and eluted in EB buffer, prior to being sheared using a Covaris M220 shearing instrument to a target size of 150 bp.The sheared product was size selected via AMPure XP to an average size of ~180bp. Synthetic spike-in control DNA waws obtained through commercial means (IDT)
The libraries were prepared using the Kapa Hyper Prep Kit using IDT xGen Stubby adapters.

tag: experiment

value: Assessing batch effects

value: AML - plasma

value: output

tag: molecule subtype

value: cfDNA


title: A29_DT_0.01ng of synthetic DNA in 10ng of AML sample: Patient 1

description:
AML plasma + synthetic spike-in DNA
Multiple identifiers for this sample are: 6654, A29, A29_DT, Sample 1, Princess Margaret Cancer Biobank ID 150279


title: A35_DT_0.01ng of synthetic DNA in 10ng of AML sample: Patient 2

description:
AML plasma + synthetic spike-in DNA
Multiple identifiers for this sample are: 6655, A35, A35_DT, Sample 2, Princess Margaret Cancer Biobank ID 151050


title: 6425_Synthetic DNA only. Input.

description:
Synthetic DNA only. Input.
6425

organism: synthetic construct

source_name: Synthetic spike-in control DNA

value: Asssessing technical bias (Figure 1)

value: synthetic

value: spike-in DNA


title: 6427_Synthetic DNA only. Output.

description:
Synthetic DNA only. Output.
6427

value: input


title: A56_DT_0.01ng of synthetic DNA in 10ng of AML sample: Patient 4

description:
AML plasma + synthetic spike-in DNA
160326


title: A64_0.01ng of synthetic DNA in 10ng of AML sample: Patient 5


title: HepG2_SmallRNA_Demethylated-rep1

description:
cell culture
Sample 3
DAMM-seq_mt-tRNA_AllMethyl.xlsx


title: 6428_Synthetic DNA only. Output.

description:
Synthetic DNA only. Output.
6428


title: A37_0.01ng of synthetic DNA in 10ng of AML sample: Patient 3

description:
AML plasma + synthetic spike-in DNA
Multiple identifiers for this sample are: 6656, A37, A37_DT, Sample 3, Princess Margaret Cancer Biobank ID 160537


title: A29_0.01ng of synthetic DNA in 10ng of AML sample: Patient 1


title: 6655_0.01ng of synthetic DNA in 10ng of AML sample: Patient 2


title: A35_0.01ng of synthetic DNA in 10ng of AML sample: Patient 2


title: A56_0.01ng of synthetic DNA in 10ng of AML sample: Patient 4


title: 6657_0.01ng of synthetic DNA in 10ng of AML sample: Patient 4

description:
AML plasma + synthetic spike-in DNA
Multiple identifiers for this sample are: 6657, A56, A56_DT, Sample 4, Princess Margaret Cancer Biobank ID 160326


title: 6658_0.01ng of synthetic DNA in 10ng of AML sample: Patient 5


title: 6547_0.01ng of synthetic DNA. 10ng of HCT116.

description:
0.01ng of synthetic DNA. 10ng of HCT116.
6547

source_name: HCT116 + synthetic spike-in DNA

value: Optimizing synthetic DNA amount (Figure 1)

value: colorectal cancer cell line (HCT116)

value: genomic DNA


title: 6656_0.01ng of synthetic DNA in 10ng of AML sample: Patient 3


title: 6654_0.01ng of synthetic DNA in 10ng of AML sample: Patient 1


title: A37_DT_0.01ng of synthetic DNA in 10ng of AML sample: Patient 3


title: 6544_0.1ng of synthetic DNA. 10 ng of HCT116.

description:
0.1ng of synthetic DNA. 10 ng of HCT116.
6544


title: 6426_Synthetic DNA only. Input.

description:
Synthetic DNA only. Input.
6426


title: KO1

status: Public on Jan 02 2021

first: Thomas

last: Ciucci

description: H-2Db-GP33+ CD8+

data_processing:
Basecalling was performed using RTA 2.4.11
Sequencing reads were demultiplexed using Bcl2fastq 2.17
10x data for scRNA samples are run through cellranger v2.2.0  to produce the fastqfiles
The fastq files are processed throgh the cellranger count to get differential expression counts.
Genome_build: mm10

source_name: Spleen

extract_protocol:
Single Cell 3 Reagent Kits v2 per the manufacturer instructions (10X genomics)
Libraries were pooled and  sequenced with paired-end reads of 26x98bp  on a NextSeq500 sequencer (Illumina).

tag: strain background

tag: mouse origin

value: Chimera 1


title: 6548_0.01ng of synthetic DNA. 10ng of HCT116.

description:
0.01ng of synthetic DNA. 10ng of HCT116.
6548


title: 6546_0.05ng of synthetic DNA. 10ng of HCT116.

description:
0.05ng of synthetic DNA. 10ng of HCT116.
6546


title: WT2

description: H-2Db-GP33+ CD8+

value: WT

value: Chimera 2


title: 6545_0.05ng of synthetic DNA. 10ng of HCT116.

description:
0.05ng of synthetic DNA. 10ng of HCT116.
6545


title: mouse E8.5 control embryo, replicate 2, 3 pooled embryos [InUt_E85_pool-3emb]

status: Public on Feb 15 2021

city: Rehovot

first: Noa

last: Novershtern

address: Weizmann Institute

department: Molecular Genetics

country: Israel

institute: Weizmann Institute of Science

description:
InUt_E85_pool-3emb
processed data file:
scRNA.normalized.assay85.csv.gz
scRNA.MetaData.85.csv.gz
scRNA.integrated.scaled.assay85.csv.gz

data_processing:
Demultiplexing was done with cellranger/3.1.0 mkfastq
Alignment and quantification of single cell features was done with cellranger counts
Aggregation of multiple GEM wells was done using-  cellranger aggr
Cells with under 200 expressing genes, over 4000 expressing genes or over 10% mitochondrial gene expression were filtered out for the rest of the analysis
Genome_build: mm10
Supplementary_files_format_and_content: proccessed data is divided into three different matrices created using Seurat 3.0: Normalized - contains normalized counts of all genes across all cells. MetaData - contains information of each of the cells, and Intagrated-  with anchors the two in-utero &ex-utero matrixes bases on the top 2000 most variable genes

source_name: E8.5 mouse embryo

extract_protocol:
Embryos were dissociated into single cells at a concentration of 1000 cells per microliter and processed in the 10X Chromium Single Cell 3ʹ v3 platform
RNA libraries were prepared with the Chromium Single Cell 3ʹ v3 by following the manufacturer instructions

tag: embryo growth conditions

value: in utero

tag: embryonic day


title: mouse E8.5 control embryo, replicate 1 [InUt_E85_1]

description:
InUt_E85_1
processed data file:
scRNA.normalized.assay85.csv.gz
scRNA.MetaData.85.csv.gz
scRNA.integrated.scaled.assay85.csv.gz


title: KO2

description: H-2Db-GP33+ CD8+


title: Liver, Hlf+/+/Dbp+/+/Tef+/+_AL_ZT12 (S3)

status: Public on Dec 03 2020

city: Lausanne

first: Benjamin

middle: D

last: Weger

state: VD

address: Station 15

department: SV

country: Germany

institute: EPFL

data_processing:
Sequences were aligned to the full reference genome index build with Ensembl annotation to improve accuracy on splices junctions using STAR 2.4.0i
To count uniquely mapped reads per gene we were using the --quantMode GeneCounts option of STAR.
Genome_build: mm10
Supplementary_files_format_and_content: raw counts

source_name: liver

extract_protocol:
Liver RNAs were extracted as previously described (Gachon et al., 2006) with classical methods using guanidium thiocyanate lysis buffer.
Libraries were prepared using the TruSeq Stranded mRNA Kit (Illumina) following the manufacturer’s protocol.

tag: feeding regimen

value: AL

tag: zeitgeber time

tag: age

value: 12-16 weeks

value: liver


title: Liver, Hlf+/+/Dbp+/+/Tef+/+_AL_ZT12 (S27)

tag: genotype


title: Liver, Hlf+/+/Dbp+/+/Tef+/+_AL_ZT04 (S1)


title: 6543_0.1ng of synthetic DNA. 10 ng of HCT116.

description:
0.1ng of synthetic DNA. 10 ng of HCT116.
6543


title: Liver, Hlf+/+/Dbp+/+/Tef+/+_AL_ZT24 (S6)


title: Liver, Hlf+/+/Dbp+/+/Tef+/+_AL_ZT04 (S25)


title: Liver, Hlf+/+/Dbp+/+/Tef+/+_AL_ZT24 (S30)


title: Liver, Hlf+/+/Dbp+/+/Tef+/+_AL_ZT16 (S28)


title: Liver, Hlf+/+/Dbp+/+/Tef+/+_AL_ZT16 (S4)


title: Liver, Hlf-/-/Dbp-/-/Tef-/-_AL_ZT16 (S34)


title: Liver, Hlf+/+/Dbp+/+/Tef+/+_AL_ZT08 (S2)


title: Liver, Hlf-/-/Dbp-/-/Tef-/-_AL_ZT04 (S13)


title: Liver, Hlf+/+/Dbp+/+/Tef+/+_AL_ZT20 (S29)


title: Liver, Hlf+/+/Dbp+/+/Tef+/+_AL_ZT08 (S26)


title: Liver, Hlf-/-/Dbp-/-/Tef-/-_AL_ZT24 (S18)


title: Liver, Hlf-/-/Dbp-/-/Tef-/-_AL_ZT24 (S36)


title: Liver, Hlf-/-/Dbp-/-/Tef-/-_AL_ZT20 (S17)


title: Liver, Hlf-/-/Dbp-/-/Tef-/-_AL_ZT16 (S16)


title: Liver, Hlf-/-/Dbp-/-/Tef-/-_AL_ZT20 (S35)


title: Liver, Hlf-/-/Dbp-/-/Tef-/-_AL_ZT12 (S15)


title: WT1

description: H-2Db-GP33+ CD8+


title: Liver, CRY_WT9_RF_RNA_zt16

extract_protocol:
Liver RNAs were extracted as previously described (Gachon et al., 2006) with classical methods using guanidium thiocyanate lysis buffer.
Libraries were prepared using the TruSeq Stranded mRNA Kit (Illumina) following the manufacturer’s protocol. We used 250 ng total RNA as starting material and performed 11 PCR cycles from library amplification (optimal cycle determined with the same method as the small RNA, see above). 96 samples were pooled, denatured, spiked with 3% PhiX and loaded onto a paired-end read flow cell v3 at a final concentration of 11 pM for a paired-end 100 cycles run, strictly following Illumina’s recommendations. Sequencing depth was equivalent to 6 samples per lane.

value: RF


title: Liver, CRY_WT9_RF_RNA_zt04


title: Liver, CRY_WT9_RF_RNA_zt00


title: Liver, Hlf-/-/Dbp-/-/Tef-/-_AL_ZT12 (S33)


title: Liver, CRY_WT6_RF_RNA_zt00


title: Liver, Hlf-/-/Dbp-/-/Tef-/-_AL_ZT08 (S14)


title: Liver, Hlf-/-/Dbp-/-/Tef-/-_AL_ZT08 (S32)


title: Liver, CRY_WT6_RF_RNA_zt04


title: Liver, Hlf+/+/Dbp+/+/Tef+/+_AL_ZT20 (S5)


title: Liver, CRY_WT6_RF_RNA_zt12


title: Liver, CRY_WT4_AL_RNA_zt16


title: Liver, CRY_WT9_RF_RNA_zt20


title: Liver, Hlf-/-/Dbp-/-/Tef-/-_AL_ZT04 (S31)


title: Liver, CRY_WT9_RF_RNA_zt08


title: Liver, CRY_WT9_RF_RNA_zt12


title: Liver, CRY_WT4_AL_RNA_zt20


title: Liver, CRY_WT6_RF_RNA_zt20


title: Liver, CRY_WT4_AL_RNA_zt04


title: Liver, CRY_WT6_RF_RNA_zt16


title: Liver, CRY_WT6_RF_RNA_zt08


title: Liver, CRY_WT2_AL_RNA_zt12


title: Liver, CRY_WT4_AL_RNA_zt12


title: Liver, CRY_WT2_AL_RNA_zt04


title: Liver, CRY_WT4_AL_RNA_zt08


title: Liver, CRY_WT2_AL_RNA_zt16


title: Liver, CRY_WT2_AL_RNA_zt08


title: Liver, CRY_WT4_AL_RNA_zt00


title: Liver, CRY_WT2_AL_RNA_zt00


title: Liver, CRY_KO_1_AL_RNA_zt08


title: Liver, CRY_KO_1_AL_RNA_zt16


title: Liver, CRY_KO_1_AL_RNA_zt12


title: Liver, CRY_KO_1_AL_RNA_zt20


title: Liver, CRY_KO_1_AL_RNA_zt04


title: Liver, CRY_WT2_AL_RNA_zt20


title: Liver, CRY_KO5_RF_RNA_zt16


title: Liver, CRY_KO5_RF_RNA_zt04


title: Liver, CRY_KO_1_AL_RNA_zt00


title: Liver, CRY_KO5_RF_RNA_zt00


title: Liver, CRY_KO3_RF_RNA_zt20


title: Liver, CRY_KO5_RF_RNA_zt20


title: Liver, CRY_KO5_RF_RNA_zt08


title: Liver, CRY_KO3_RF_RNA_zt16


title: Liver, CRY_KO3_RF_RNA_zt00


title: Liver, CRY_KO3_RF_RNA_zt04


title: Liver, CRY_KO3_RF_RNA_zt12


title: Liver, CRY_KO3_RF_RNA_zt08


title: Liver, CRY_KO2_AL_RNA_zt20


title: Liver, CRY_KO2_AL_RNA_zt08


title: Liver, CRY_KO2_AL_RNA_zt16


title: Liver, CRY_KO2_AL_RNA_zt12


title: Liver, CRY_KO2_AL_RNA_zt04


title: Liver, CRY_KO2_AL_RNA_zt00


title: Liver, Bmal1_WT8_AL_RNA_zt20

description: Bmal1  WT_AL_ZT20

value: Bmal1  WT


title: Liver, Bmal1_WT8_AL_RNA_zt08

description: Bmal1  WT_AL_ZT08


title: Liver, Bmal1_WT8_AL_RNA_zt16

description: Bmal1  WT_AL_ZT16


title: Liver, CRY_KO5_RF_RNA_zt12


title: Liver, Bmal1_WT8_AL_RNA_zt04

description: Bmal1  WT_AL_ZT04


title: Liver, Bmal1_WT4_RF_RNA_zt16

description: Bmal1  WT_RF_ZT16


title: Liver, Bmal1_WT4_RF_RNA_zt20

description: Bmal1  WT_RF_ZT20


title: Liver, Bmal1_WT4_RF_RNA_zt12

description: Bmal1  WT_RF_ZT12


title: Liver, Bmal1_WT8_AL_RNA_zt00

description: Bmal1  WT_AL_ZT00


title: Liver, Bmal1_WT4_RF_RNA_zt08

description: Bmal1  WT_RF_ZT08


title: Liver, Bmal1_WT4_RF_RNA_zt00

description: Bmal1  WT_RF_ZT00


title: Liver, Bmal1_WT2_RF_RNA_zt16

description: Bmal1  WT_RF_ZT16


title: Liver, Bmal1_WT2_RF_RNA_zt20

description: Bmal1  WT_RF_ZT20


title: Liver, Bmal1_WT4_RF_RNA_zt04

description: Bmal1  WT_RF_ZT04


title: Liver, Bmal1_WT2_RF_RNA_zt12

description: Bmal1  WT_RF_ZT12


title: Liver, Bmal1_WT1_AL_RNA_zt20

description: Bmal1  WT_AL_ZT20


title: Liver, Bmal1_WT1_AL_RNA_zt16

description: Bmal1  WT_AL_ZT16


title: Liver, Bmal1_WT2_RF_RNA_zt00

description: Bmal1  WT_RF_ZT00


title: Liver, Bmal1_WT1_AL_RNA_zt08

description: Bmal1  WT_AL_ZT08


title: Liver, Bmal1_WT1_AL_RNA_zt00

description: Bmal1  WT_AL_ZT00


title: Liver, Bmal1_WT1_AL_RNA_zt12

description: Bmal1  WT_AL_ZT12


title: Liver, Bmal1_WT1_AL_RNA_zt04

description: Bmal1  WT_AL_ZT04


title: Liver, Bmal1_KO8_AL_RNA_zt16

description: Bmal1 KO_AL_ZT16

value: Bmal1 KO


title: Liver, Bmal1_WT8_AL_RNA_zt12

description: Bmal1  WT_AL_ZT12


title: Liver, Bmal1_KO8_AL_RNA_zt04

description: Bmal1 KO_AL_ZT04


title: Liver, Bmal1_KO8_AL_RNA_zt00

description: Bmal1 KO_AL_ZT00


title: Liver, Bmal1_KO4_RF_RNA_zt16

description: Bmal1 KO_RF_ZT16


title: Liver, Bmal1_KO8_AL_RNA_zt08

description: Bmal1 KO_AL_ZT08


title: Liver, Bmal1_KO4_RF_RNA_zt12

description: Bmal1 KO_RF_ZT12


title: Liver, Bmal1_KO4_RF_RNA_zt08

description: Bmal1 KO_RF_ZT08


title: Liver, Bmal1_KO4_RF_RNA_zt04

description: Bmal1 KO_RF_ZT04


title: Liver, Bmal1_KO4_RF_RNA_zt00

description: Bmal1 KO_RF_ZT00


title: Liver, Bmal1_WT2_RF_RNA_zt08

description: Bmal1  WT_RF_ZT08


title: Liver, Bmal1_KO2_RF_RNA_zt16

description: Bmal1 KO_RF_ZT16


title: Liver, Bmal1_KO2_RF_RNA_zt20

description: Bmal1 KO_RF_ZT20


title: Liver, Bmal1_WT2_RF_RNA_zt04

description: Bmal1  WT_RF_ZT04


title: Liver, Bmal1_KO2_RF_RNA_zt00

description: Bmal1 KO_RF_ZT00


title: Liver, Bmal1_KO2_RF_RNA_zt12

description: Bmal1 KO_RF_ZT12


title: Liver, Bmal1_KO1_AL_RNA_zt16

description: Bmal1 KO_AL_ZT16


title: Liver, Bmal1_KO1_AL_RNA_zt20

description: Bmal1 KO_AL_ZT20


title: Liver, Bmal1_KO8_AL_RNA_zt12

description: Bmal1 KO_AL_ZT12


title: Liver, Bmal1_KO2_RF_RNA_zt08

description: Bmal1 KO_RF_ZT08


title: Liver, Bmal1_KO2_RF_RNA_zt04

description: Bmal1 KO_RF_ZT04


title: Liver, Bmal1_KO8_AL_RNA_zt20

description: Bmal1 KO_AL_ZT20


title: HK7

status: Public on Nov 17 2020

first: Jorge

last: Moscat

address: 1188 York Avenue

department: Pathology and Laboratory Medicine

institute: WEILL CORNELL MEDICINE

description:
HK7_S1
HK10_S1

data_processing:
Raw data was downloaded in Ubuntu using BaseSpaceCLI.
We used cellranger mkfastq to demultiplex raw base call (BCL) files generated by Illumina sequencers into FASTQ files
We used cellranger count to take FASTQ files from cellranger mkfastq and perform alignment to mm10, filtering, barcode counting, and UMI counting
We used cellranger aggr to aggregate both sample outputs of cellranger count, normalizing  to the same sequencing depth and then recomputing the feature-barcode matrices and analysis on the combined data
We used Seurat in R to analyzes the aggregated data
Genome_build: mm10
Supplementary_files_format_and_content: HDF5 file containing per-molecule information for all molecules that contain a valid barcode and valid UMI and were assigned with high confidence to a gene or Feature Barcode. This HDF5 file contains data corresponding to the observed molecules, as well as data about the libraries, feature set(s), and barcode lists used for the analysis

source_name: Orthotopic tumor

growth_protocol: MTOs (equivalent to 5 x 105 cells) and fibroblasts (5 x 10^5 cells) were injected in cecum orthotopically. Four weeks after injection, tumors were harvested.

extract_protocol:
Tumors were minced thoroughly and digested by 0.5mg/ml Liberase TH (Sigma) for 30 minutes at 37°C. Dead cells were removed by Annexin V (STEMCELL technologies).
scRNA-seq libraries were generated using the Chromium Single Cell 30 Reagent Kit v2 (10X Genomics). Cells were loaded onto the 10X Chromium Single Cell Platform (10X Genomics) at a concentration of 2,000 cells per µl (Single Cell 3′ library and Gel Bead Kit v.2) as described in the manufacturer’s protocol (10x User Guide, Revision B). On average, approximately 8,000 cells were loaded. Generation of gel beads in emulsion (GEMs), barcoding, GEM-RT clean-up, complementary DNA amplification and library construction were all performed as per the manufacturer’s protocol. Individual sample quality was checked using a Bioanalyzer Tapestation (Agilent). Qubit was used for library quantification before pooling. The final library pool was sequenced on an Illumina NovaSeq 6000 instrument using a S1 flowcell. Average cell recovery was 23,576 cells, with a total of 47153 cells captured at a mean depth of 18712 reads per cell and 1224 mean genes per cell.

tag: genotype/variation

value: WT sgScr

value: 12 weeks old

value: colon

tag: tissue type

value: Orthotopic tumor


title: HK8

description:
HK8_S2
HK11_S2

value: Prkcz-/- sgScr

tag: tissue


title: HK9

description:
HK9_S3
HK12_S3

value: Prkcz-/- sgSfrp2


title: Liver, Bmal1_KO4_RF_RNA_zt20

description: Bmal1 KO_RF_ZT20


title: Liver, Bmal1_KO1_AL_RNA_zt04

description: Bmal1 KO_AL_ZT04


title: HK1

description:
WT
filtered_feature_bc_matrix.h5

data_processing:
Raw data was downloaded in Ubuntu using BaseSpaceCLI.
We used cellranger mkfastq to demultiplex raw base call (BCL) files generated by Illumina sequencers into FASTQ files
We used cellranger count to take FASTQ files from cellranger mkfastq and perform alignment to mm10, filtering, barcode counting, and UMI counting
We used cellranger aggr to aggregate both sample outputs of cellranger count, normalizing  to the same sequencing depth and then recomputing the feature-barcode matrices and analysis on the combined data
We used Seurat in R to analyzes the aggregated data
Genome_build: mm10
Supplementary_files_format_and_content: filtered_feature_bc_matrix.h5
Supplementary_files_format_and_content: HDF5 file containing per-molecule information for all molecules that contain a valid barcode and valid UMI and were assigned with high confidence to a gene or Feature Barcode. This HDF5 file contains data corresponding to the observed molecules, as well as data about the libraries, feature set(s), and barcode lists used for the analysis

growth_protocol: MTOs (equivalent to 5 x 105 cells) were injected in rectum orthotopically. Four weeks after injection, tumors were harvested.

extract_protocol:
Tumors were minced thoroughly and digested by 0.5mg/ml Liberase TH (Sigma) for 30 minutes at 37°C. Dead cells were removed by Annexin V (STEMCELL technologies).
scRNA-seq libraries were generated using the Chromium Single Cell 30 Reagent Kit v2 (10X Genomics). Cells were loaded onto the 10X Chromium Single Cell Platform (10X Genomics) at a concentration of 2,000 cells per µl (Single Cell 3′ library and Gel Bead Kit v.2) as described in the manufacturer’s protocol (10x User Guide, Revision B). On average, approximately 8,000 cells were loaded. Generation of gel beads in emulsion (GEMs), barcoding, GEM-RT clean-up, complementary DNA amplification and library construction were all performed as per the manufacturer’s protocol. Individual sample quality was checked using a Bioanalyzer Tapestation (Agilent). Qubit was used for library quantification before pooling. The final library pool was sequenced on an Illumina NovaSeq6000 instrument using a S1 flowcell. Average cell recovery was 23,576 cells, with a total of 47153 cells captured at a mean depth of 18712 reads per cell and 1224 mean genes per cell.

tag: strain

value: Prkczfl/fl


title: Liver, Bmal1_KO1_AL_RNA_zt00

description: Bmal1 KO_AL_ZT00


title: HK2

description:
PKCzKO
filtered_feature_bc_matrix.h5


title: zKO

description: PKCzKO

data_processing:
FASTQ.gz files were concatenated into one file per sample and allignment to mm10 was done using bowtie2 to generate .bam files
We used DeepTools to remove duplicates and do bamCoverage to generate bigwig (.bw) files from .bam files.
We called peaks using MACS2.
We visualized peaks in IGV.
Genome_build: mm10
Supplementary_files_format_and_content: processed data file consist of a bigwig file per sample

source_name: colon fibroblasts

growth_protocol: Colon fibroblasts were cultured in Dulbecco's Modified Eagle Medium (DMEM, Corning), supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and 100 U/ml penicillin and 100 µg/ml streptomycin, in an atmosphere of 95% air and 5% CO2

extract_protocol:
The cell pellet was resuspended in 50 μl lysis buffer and then spun down 500 × g for 10 min at 4 °C. The nuclei pellet was resuspended into 50 μl transposition reaction mixture containing Tn5 transposase from Nextera DNA Library Prep Kit (Illumina) and incubated at 37 °C for 30 min. Then the transposase-associated DNA was purified using MinElute PCR purification kit (QIAGEN). To amplify the library, the DNA was first amplified for 5 cycles using indexing primer from Nextera kit and NEBNext High-Fidelity 2X PCR master mix. To reduce the PCR amplification bias, 5 μl of amplified DNA after the first 5 cycles was used to do qPCR of 20 cycles to decide the number of cycles for the second round of PCR.
The total amplified DNA was size selected to fragments less than 800 bp using SPRI beads. Quantification of the ATAC-seq library was done with QuBit. The size of the pooled library was examined by TapeStation. Barcoded ATAC-seq libraries were pooled and paired end sequenced (sX75) on the Illumina NextSeq 500 using the High output V2.5 kit (Illumina Inc., San Diego, CA).

treatment_protocol: No treatment was applied. Cells were harvested at ~80% confluency 24h after plating.

value: Prkcz-/-


title: PKCZKO_sgSox2_2

description:
PKCz/Sox2DKO
GeneMatrix_depthnormalized_quantseq.xlsx

data_processing:
Read data was processed with the BlueBee Genomics Platform (BlueBee, San Mateo, CA).
Reads were aligned using Lexogen Quantseq 2.3.1 FWD UMI pipeline, and differential transcript expression was determined using Lexogen QuantSeq DE 1.3.1 pipeline. Both pipelines are from BluBee Genomics Platform.
Counts were normalized by total number of reads per each samples and multiplied by 10E6.
Genes with 0 counts in all the samples were removed.
Genome_build: Mus musculus - mouse (GRCm38)
Supplementary_files_format_and_content: Counts matrix with gene expression value for all samples.

extract_protocol:
RNA was harvested using Trizol reagent and purified using Quick-RNA MiniPrep kit (Zymo Research).
Libraries were prepared from 200 ng of total RNA using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina from Lexogen, and optional UMIs (Vienna, Austria). Barcoded libraries were pooled, and single end sequenced (1X75) on the Illumina NextSeq 500 using the High output V2.5 kit (Illumina Inc., San Diego CA).

value: Prkcz-/- sgSox2


title: PKCZKO_sgSox2_4


title: PKCZKO_sgSox2_3


title: PKCZKO_sgSox2_1


title: PKCZKO_sgScr_4

description:
PKCzKO
GeneMatrix_depthnormalized_quantseq.xlsx


title: WT

description: WT (wild type)

value: WT


title: Liver, Bmal1_KO1_AL_RNA_zt12

description: Bmal1 KO_AL_ZT12


title: PKCZKO_sgScr_2


title: WT_sgScr_4

description:
WT
GeneMatrix_depthnormalized_quantseq.xlsx


title: PKCZKO_sgScr_1


title: Liver, Bmal1_KO1_AL_RNA_zt08

description: Bmal1 KO_AL_ZT08


title: WT_sgScr_3


title: PKCZKO1_03

description:
PKCzKO
ZKO_vs_WT.FPKM_delete_zero.xlsx

data_processing:
FASTq files were processed using RNAseq Alignment module in BaseSpace (Illumina), using default parameters. Reads were aligned to mouse (mm10) genome using STAR. Differentially expressed genes were calculated using DeSeq2.
Genome_build: mm10
Supplementary_files_format_and_content: FPKM gene expression values for all mouse genes (mm10) for all samples

extract_protocol:
RNA was harvested using Trizol reagent and purified using Quick-RNA MiniPrep kit (Zymo Research).
PolyA RNA was isolated using the NEBNext® Poly(A) mRNA Magnetic Isolation Module and barcoded libraries were made using the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, Ipswich MA). Libraries were pooled and single end sequenced (1X76) on the Illumina NextSeq 500 using the High output V2 kit (Illumina Inc., San Diego CA).


title: WT_sgScr_1


title: WT_sgScr_2


title: PKCZKO1_02

description:
PKCzKO
ZKO_vs_WT.FPKM_delete_zero.xlsx


title: PKCZKO1_01

description:
PKCzKO
ZKO_vs_WT.FPKM_delete_zero.xlsx


title: WT1_01

description:
WT (wild type)
ZKO_vs_WT.FPKM_delete_zero.xlsx


title: WT1_03


title: WT1_02


title: PKCZKO_sgScr_3


title: ABC1-vehicle_1

status: Public on Sep 23 2020

city: Sendai

first: Hozumi

last: Motohashi

address: 2-1 Seiryo-machi, Aoba-ku

department: Medical Biochemistry

country: Japan

institute: Tohoku University

data_processing:
Illumina bcl2fastq v2.15.0 software was used for basecalling.
Possible poly-A tails were trimmed using the cutadapt (version 1.15 with Python 3.5.2) with parameters -a "A{100}"
Reads were mapped using the STAR (version 2.5.3a) with parameters --outSAMattrIHstart 0 --outSAMstrandField intronMotif --outSAMtype BAM SortedByCoordinate. Genome indices are generated upon the hg19 genome assembly and the GENCODE release 19 (GRCh37.p13) annotation.
BAM files for each samples from different lanes are merged into single BAM files using SAMTools merge version 1.3.1.
Cuffquant and Cuffnorm were used to obtain FPKM values for each GENCODE annotated genes. Cuffquant (version v2.2.1) was performed using parameters --library-type fr-firststrand and a mask file including mtRNAs and rRNAs described in the annotation. Cuffnorm (version v2.2.1) was used with parameters --library-type fr-firststrand to obtain normalized expression values.
HT-Seq and DESeq2 were used to perform differential expression analysis. Raw read counts for each annotated genes were obtained using htseq-count (HTSeq framework version 0.9.1) with parameters -s reverse. DESeq2 (R version 3.2.0, package version 1.8.2) was used with default parameters to obtain differential expression profiles for each cell types.
Genome_build: hg19
Supplementary_files_format_and_content: processed data file: Tab-delimited text files include FPKM values for each annotated genes from a Cuffnorm genes.fpkm_table file. supplementary file: Tab-delimited text files generated by DESeq2 for each cell types.

source_name: ABC1 cells

growth_protocol: ABC1 and HCC4006 cells were cultured in DMEM (low glucose) containing 10% fetal bovine serum.

extract_protocol:
Total RNA was purified using RNeasy Mini Kit (Qiagen) and used for preparation of cDNA sequencing libraries.
cDNA sequencing libraries were constructed employing SureSelect Strand-Specific RNA library preparation kit (Agilent Technologies) after the polyA selection step.

treatment_protocol: ABC1 and HCC4006 cells were treated with 100-μM DEM for 16 hours.

tag: cell line

value: No mutations in KEAP1 or NRF2 gene

tag: condition

value: vehicle treated

tag: cell type

value: lung adenocarcinoma


title: HCC4006-vehicle_0

source_name: HCC4006 cells


title: HCC4006-DEM_0

value: DEM treated


title: HCC4006-DEM_1


title: ABC1-DEM_1


title: HCC4006-vehicle_1


title: H460-ctrl_0

data_processing:
Illumina bcl2fastq v2.15.0 software was used for basecalling.
Possible poly-A tails were trimmed using the cutadapt (version 1.15 with Python 3.5.2) with parameters -a "A{100}"
Reads were mapped using the STAR (version 2.5.3a) with parameters --outSAMattrIHstart 0 --outSAMstrandField intronMotif --outSAMtype BAM SortedByCoordinate. Genome indices are generated upon the hg19 genome assembly and the GENCODE release 19 (GRCh37.p13) annotation.
BAM files for each sample from different lanes are merged into single BAM files using SAMTools merge version 1.3.1.
Cuffquant and Cuffnorm were used to obrain FPKM values for each GENCODE annotated gene. Cuffquant (version v2.2.1) was performed using parameters --library-type fr-firststrand and a mask file including mtRNAs and rRNAs described in the annotation. Cuffnorm (version v2.2.1) was used with parameters --library-type fr-firststrand to obtain normalized expression values.
HT-Seq and DESeq2 were used to perform differential expression analysis. Raw read counts for each annotated genes were obtained using htseq-count (HTSeq framework version 0.9.1) with parameters -s reverse. DESeq2 (R version 3.2.0, package version 1.8.2) was used with default parameters to obtain differential expression profiles for each cell types.
Genome_build: hg19
Supplementary_files_format_and_content: processed data file: Tab-delimited text files include FPKM values for each annotated gene from a Cuffnorm genes.fpkm_table file. supplementary file: Tab-delimited text files generated using DESeq2 for each cell types.

growth_protocol: A549 cell were cultured in DMEM (high glucose) containing 10% fetal bovine serum, and H460 and H2023 cells were cultured in DMEM (low glucose) containing 10% fetal bovine serum.

extract_protocol:
Total RNA was purified using RNeasy Mini Kit (Qiagen).
For the analysis of A549 cells, 4.0 μg of total RNA was subjected to the removal of rRNA by Ribo-Zero Gold kit (Illumina) and used for preparation of cDNA sequencing libraries employing SureSelect Strand-Specific RNA library preparation kit (Agilent Technologies) with a modified protocol omitting the polyA selection step. For the analyses of H460 and H2023 cells, total RNA was used for preparation of cDNA sequencing libraries employing SureSelect Strand-Specific RNA library preparation kit (Agilent Technologies) after the polyA selection step.

treatment_protocol: siRNA against NRF2 or control siRNA was transfected to A549, H2023 and H460 cells, and the cells were harvested for RNA purification after 48 hours.

value: KEAP1 mutation positive

value: control siRNA introduced

value: lung large cell carcinoma


title: ABC1-vehicle_0


title: H460-KD_1

value: NRF2 siRNA '#30' introduced


title: H460-ctrl_1


title: A549-ctrl_1


title: ChIP-seq_A549-input_2

data_processing:
Illumina bcl2fastq v2.15.0 software used for basecalling.
To trim adapters, cutadapt (version 1.15 with Python 2.7.10) was used with parameters -b file:truseq_adapters.fasta -m 25
Reads were aligned to the hg19 using Bowtie2 v2.2.6 with default parameters.
For each sample, BAM files are merged into single BAM files using SAMTools merge (version 1.3.1).
Reads that have mapping quality less than 20 were removed using SAMTools view (version 1.3.1.) with arguments -bh -q 20
Peaks were called using MACS2 v2.1.0.20151222 with parameters -g hs --broad --bdg. For each treat data, DNA input data with a same condition (control or NRF2 siRNA introduced) were used as control BAM.
Peaks overlapped with regions described in the ENCODE blacklist for hg19 (obtained on March 22, 2016) are filtered out using bedtools intersect (version 2.26.0).
To obtain H3K27ac modification levels, treat_pileup.bdg files from MACS2 were used as occupancy profiles. Three bedgraph files for each condition were merged into single wig files using WiggleTools mean command (version 1.2). Wig files were converted into BigWig files using UCSC wigToBigWig. Before mean calculation, each wig files were normalized by total number of mapped reads using WiggleTools scale command.
Genome_build: hg19
Supplementary_files_format_and_content: Bed files were generated using MACS2. supplementary file: BigWig files for each condition contain mean of depth of coverage for 3 samples. The depth is normalized as per 1 million mapped reads.

growth_protocol: A549 cells were cultured in DMEM (high glucose) containing 10% fetal bovine serum.

extract_protocol:
The cells were cross-linked by treatment with 1% formaldehyde for 10 min. The samples were then lysed and sonicated to shear DNA. Chromatin immunoprecipitation was performed with anti-H3K27ac antibody (MABI0309; Cosmo Bio). Precipitated DNA was de-crosslinked, purified and used for library preparation.
Sequencing libraries were prepared from 1.0 or 2.0 ng of ChIPed DNA and input samples by a Mondrian SP+ system (Nugen) with an Ovation SP Ultralow DR Multiplex System (Nugen). The libraries were further purified and size-selected using an AMPure XP Kit (Beckman Coulter) and were quantified by a quantitative MiSeq (qMiSeq) method.

treatment_protocol: NRF2 siRNA or control siRNA was transfected to A549 cells, and the cells were harvested for chromatin immunoprecipitation after 48 hours. Two alternative NRF2 siRNAs were used, #28 and #30.

tag: chip-antibody

value: none (DNA input)

value: NRF2 siRNA '30' introduced


title: ABC1-DEM_0


title: H460-KD_0

value: NRF2 siRNA '#28' introduced


title: ChIP-seq_A549-ctrl_3

value: MABI0309; Cosmo Bio


title: A549-KD_1


title: H2023-KD_1


title: H2023-ctrl_0


title: H2023-KD_0


title: ChIP-seq_A549-input_1


title: A549-KD_0


title: Nac_mCRS_16

status: Public on Sep 20 2020

data_processing:
Base-calls were performed with Illumina bcl2fastq
fastq files were demultiplexed using BRBseq_Tools suite (https://github.com/DeplanckeLab/BRB-seqTools)
reads were aligned using STAR (version 020201) (Dobin et al 2013), and count matrices were generated with HTSeq (version 0.9.1) (Love et al 2014).
Genome_build: mm10

source_name: nucleus accumbens

extract_protocol: RNA sequencing libraries were prepared using 50 ng of total RNA samples following BRB-seq protocol (Alpern et al., 2019). Briefly, each RNA sample was reverse transcribed in a 96-well plate using SuperScriptTM II Reverse Transcriptase_x000B_with individual barcoded oligo-dT primers. Next, all the samples were pooled together, column purified and treated with exonuclease I. Double-stranded cDNA was generated by the second stand synthesis with DNA polymerase I following the nick translation method. Full-length double-stranded cDNA was purified with AMPure XP magnetic beads and eluted in 20 μL of water. The Illumina compatible libraries were prepared by tagmentation of 5 ng of full-length double-stranded cDNA with in-house produced Tn5 enzyme. The final library was amplified 15 cycles and the fragments ranging 200–1000 bp were size-selected using AMPure beads. The libraries were sequenced using the Illumina NextSeq 500 platform with a custom primer and the High Output v2 kit. The library loading concentration was 2.2 pM and sequencing configuration as following: R1 6c / index 8 c / R2 70c.

value: nucleus accumbens

value: 15 weeks

value: wild-type

tag: treatment

value: multimodal chronic restraint stress


title: A549-ctrl_0


title: ChIP-seq_A549-ctrl_1


title: ChIP-seq_A549-KD_3


title: ChIP-seq_A549-KD_2


title: Nac_mCRS_15


title: Nac_mCRS_11


title: ChIP-seq_A549-KD_1

value: NRF2 siRNA '28' introduced


title: Nac_CTR_16

value: control (no stress)


title: ChIP-seq_A549-ctrl_2


title: Nac_CTR_12


title: Nac_CTR_10


title: Nac_mCRS_08


title: Nac_mCRS_07


title: H2023-ctrl_1


title: Nac_mCRS_14


title: Nac_mCRS_03


title: Nac_mCRS_13


title: Nac_mCRS_09


title: Nac_CTR_08


title: Nac_mCRS_01


title: Nac_mCRS_12


title: Nac_mCRS_10


title: Nac_CTR_06


title: Nac_CTR_04


title: Nac_CTR_01


title: Nac_CTR_13


title: Nac_CTR_09


title: PFC_mCRS_13

source_name: prefrontal cortex

value: prefrontal cortex


title: Nac_mCRS_04


title: Nac_CTR_11


title: PFC_mCRS_12


title: Nac_CTR_15


title: Nac_mCRS_05


title: Nac_mCRS_06


title: Nac_mCRS_02


title: Nac_CTR_05


title: PFC_CTR_15


title: Nac_CTR_14


title: Nac_CTR_03


title: PFC_mCRS_14


title: PFC_CTR_10


title: PFC_mCRS_15


title: Nac_CTR_02


title: PFC_mCRS_10


title: Nac_CTR_07


title: PFC_mCRS_11


title: PFC_mCRS_09


title: PFC_CTR_14


title: PFC_CTR_12


title: PFC_CTR_13


title: PFC_mCRS_08


title: PFC_CTR_11


title: PFC_CTR_03


title: PFC_CTR_02


title: PFC_CTR_09


title: WT_n_3

status: Public on Sep 11 2020

data_processing:
Image analysis and base calling were performed using RTA 2.7.3 and bcl2fastq 2.17.1.14.
Sequences were aligned to the full reference genome index build with Ensembl annotation to improve accuracy on splices junctions using STAR 2.4.0i
A custom Perl script was used to quantify uniquely mapped reads. For each Ensembl gene, all exons of the respective annotated protein-coding transcripts were considered.
Genome_build: zv10
Supplementary_files_format_and_content: counts_mutants.txt: Tab-delimited text file contains raw counts.

organism: Danio rerio

source_name: total RNA

extract_protocol:
Embryos were sampled in liquid nitrogen and stored at -80°C until further processing. RNA was extracted with TRIzol (Life Technologies) following the manufacturer’s instructions with minor modifications: 30 larvae were sampled in a volume of 1.5 ml TRIzol reagent. Samples were kept at least overnight at -80°C. In order to disrupt the cell membranes, the samples were passed four times through a syringe with a diameter of 0.45 mm.
Total RNA-Seq libraries were generated from 500 ng of total RNA using TruSeq Stranded Total RNA Library Prep Human/Mouse/Rat kit and TruSeq RNA Single Indexes kits A and B (Illumina, San Diego, CA), according to manufacturer's instructions. Briefly, cytoplasmic and mitochondrial ribosomal RNA (rRNA) was removed using biotinylated, target-specific oligos combined with Ribo-Zero rRNA removal beads. Following purification, the depleted RNA was fragmented into small pieces using divalent cations at 94oC for 2 minutes. Cleaved RNA fragments were then copied into first strand cDNA using reverse transcriptase and random primers followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. Strand specificity was achieved by replacing dTTP with dUTP during second strand synthesis. The double stranded cDNA fragments were blunted using T4 DNA polymerase, Klenow DNA polymerase and T4 PNK. A single 'A' nucleotide was added to the 3' ends of the blunt DNA fragments using a Klenow fragment (3' to 5'exo minus) enzyme. The cDNA fragments were ligated to double stranded adapters using T4 DNA Ligase. The ligated products were enriched by PCR amplification (30 sec at 98oC; [10 sec at 98oC, 30 sec at 60oC, 30 sec at 72oC] x 12 cycles; 5 min at 72oC). Surplus PCR primers were further removed by purification using AMPure XP beads (Beckman-Coulter, Villepinte, France) and the final cDNA libraries were checked for quality and quantified using capillary electrophoresis.

value: embryo

tag: phenotype

value: unaffected epiboly

tag: Stage

value: 5 hpf


title: WT_n_2


title: PFC_mCRS_07


title: WT_n_1


title: PFC_mCRS_04


title: PFC_mCRS_05


title: PFC_mCRS_03


title: PFC_CTR_07


title: mondoa_n_2

value: MZmondoA mutant (allel:ka405, ZFIN ID:ZDB-ALT-180628-2)


title: PFC_mCRS_01


title: PFC_mCRS_02


title: PFC_mCRS_06


title: mondoa_n_3


title: PFC_CTR_06


title: mondoa_mo_3 [antiMon3]

data_processing:
mapping using STAR 2.3.8
Uniquely mapped reads were counted for each gene locus defined in the Ensembl 96 release. Briefly, we applied SAMtools view (Li et al., 2009) to assign uniquely mapped paired-end reads
Genome_build: GRCz11/danRer11
Supplementary_files_format_and_content: tab delimited table containing raw counts for each sample

extract_protocol:
TRIzol extraction was performed as described in the manufacturer’s protocol, with minor modifications. If not otherwise stated, 30 embryos were harvested in 1.5 ml TRIzol. Embryos were disrupted in the TRIzol solution with micropestles (Eppendorf), and 5 x 105 cells in 25 cm2 flasks were harvested with 1.5 ml TRIzol solution. Samples were then stored overnight at –80°C. After thawing the samples on ice, they were passed through a syringe four times (Braun Sterican, 0.45 x 25 mm).
Indexed mRNAseq libraries were generated from morphant samples following the TruSeq mRNA kit protocol (Illumina).
Libraries were sequenced on a HiSeq 1000 system (Illumina)  in 3 lanes of a paired-end flow cell. For each end, 50 nucleotides were sequenced. Cluster and  sequencing reactions were performed with the TruSeq SBS kit v3-HS and the paired-end cluster v3-  cBot-HS kit.

value: mondoa-mo injected

value: 4 hpf


title: PFC_CTR_05


title: mondoa_mis_2 [CtrlMo_2]

value: mondoa-mis injected


title: PFC_CTR_01


title: PFC_CTR_04


title: mondoa_s_3

description: counts_exon.txt, counts_intron.txt

data_processing:
Image analysis and base calling were performed using RTA 2.7.3 and bcl2fastq 2.17.1.14.
Sequences were aligned to the full reference genome index build with Ensembl annotation to improve accuracy on splices junctions using STAR 2.4.0i
A custom Perl script was used to quantify uniquely mapped reads. For each Ensembl gene, all exons of the respective annotated protein-coding transcripts were considered.
Genome_build: zv10
Supplementary_files_format_and_content: counts_exon.txt, exonic counts for each indicated gene and  sample

value: affected epiboly


title: mondoa_s_2

description: counts_exon.txt, counts_intron.txt


title: PFC_CTR_08


title: mondoa_s_1

description: counts_exon.txt, counts_intron.txt


title: PrkciffAlbcre_DENHFD_1

status: Public on Jun 30 2020

data_processing:
FASTQ files were processed using RNAseq Alignment module in BaseSpace (Illumina), using default parameters. Reads were aligned to mm10 genome using STAR. Differentially expressed genes were calculated using DeSeq2.
processed data file consist of an excel file matrix with gene symbols and FPKM counts for all samples
Genome_build: mm10
Supplementary_files_format_and_content: processed data file consist of an excel file matrix with gene symbols and gene counts for all samples

source_name: Prkciflox/floxAlbCre mouse under DEN/HFD protocol

extract_protocol:
Tissues were removed, flash frozen in liquid nitrogen, and RNA was extracted using TRIzol reagent (Invitrogen), and purified by using Quick-RNA Mini Prep Kit (Zymo Research) following the manufacturer’s protocols. PolyA RNA was isolated using the NEBNext Poly(A) mRNA Magnetic Isolation Module and barcoded libraries were made using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB).  Libraries were pooled and single end sequenced (1X75) on the Illumina NextSeq 500 using the High output V2.5 kit (Illumina Inc.)
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Two-week-old mice were intraperitoneally injected with hepatic carcinogen diethylnitrosamine (DEN, 25 mg/kg) and then kept with 60% fat diet as a tumor promoter for 32 weeks.

value: Prkciflox/floxAlbCre

value: liver


title: PrkciffAlbcre_DENHFD_2


title: mondoa_n_1


title: mondoa_mo_2 [antiMon2]


title: Prkciff_DENHFD_3

source_name: Prkciflox/flox mouse under DEN/HFD protocol

value: Prkciflox/flox


title: PrkciffAlbcre_RD_1

source_name: Prkciflox/floxAlbCre mouse fed with regular chow

treatment_protocol: Regular chow

tag: strain background


title: mondoa_mis_3 [CtrlMo_3]


title: mondoa_mo_1 [antiMon1]


title: PrkciffAlbcre_RD_3


title: Prkciff_RD_1

source_name: Prkciflox/flox mouse fed with regular chow


title: Adv-p62 KIR- [K1]

status: Public on Apr 28 2020

data_processing:
Quality scores for Raw reads were Sanger transformed using FASTq Groomer
FASTq Groomer outputs were aligned to mm10 genome using TopHat (-firs strand) –local sensitive
Aligned reads were sorted by coordinates using Sort BAM module
Gene Expression Estimates were calculate using Cufflinks with reference mm10 GTF file from iGenomes
Differential Gene Expression was calculated for all pairs using CuffDiff module
Genome_build: mm10
Supplementary_files_format_and_content: Gene Expression Matrix was pooled from Gene expression estimates from Cufflinks output as tab delimited file with gene count information for all samples.

source_name: Adv-p62 KIR-_lilver

extract_protocol: Livers were removed, flash frozen in liquid nitrogen, and RNA was extracted from liver tissue  using RNeasy Plus Mini kit (Qiagen Cat#74134).

tag: genotype background

value: liver specific p62 knock out

value: adenovirus-p62KIR mutant

value: overnight (15 hrs) fasting before harvesting sample


title: Adv-GFP [G3]

value: adenovirus-GFP


title: mondoa_mis_1 [CtrlMo_1]


title: PrkciffAlbcre_DENHFD_4


title: PrkciffAlbcre_DENHFD_3


title: Prkciff_DENHFD_2


title: Prkciff_DENHFD_1


title: control 3 (non adenovirus infected) [N3]


title: control 2 (non adenovirus infected) [N2]


title: FH9NRFF

growth_protocol: regular chow fed mice

extract_protocol:
Livers were removed, flash frozen in liquid nitrogen, and RNA was extracted from liver tissue using RNeasy Plus Mini kit (Qiagen Cat#74134).
Libraries were prepared from isolated total RNA using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina from Lexogen, (Vienna, Austria). Barcoded libraries were pooled and single end sequenced (1X75) on the Illumina NextSeq 500 using the High output V2.5 kit (Illumina Inc., San Diego CA).

treatment_protocol: overnight (15 hrs) fasting before harvesting sample

value: liver specific Nrf2 activated mice


title: FH10NRFF


title: Adv-p62 KIR- [K3]


title: Prkciff_RD_2


title: Prkciff_RD_3


title: Adv-p62 wild type [W2]

source_name: Adv-p62 wild type_liver

value: adenovirus-p62 wild type


title: Adv-p62 wild type [W3]


title: PrkciffAlbcre_RD_2


title: Adv-p62 KIR- [K2]


title: xAML-3_F823

status: Public on Nov 25 2019

city: Montreal

first: Sonia

last: Cellot

state: Quebec

address: 3175 Côte-Sainte-Catherine

institute: Centre Hospitalier Universitaire (CHU) Sainte-Justine Research Center

data_processing:
Basecalls performed using Illumina RTA 1.18.64
Samples demultiplexed using bcl2fastq 2.17
Sequenced reads were trimmed for sequencing adapters and low quality extrimities and mapped to the reference genome using STAR version 2.5.1b
Readcounts were computed by STAR during sequence alignment based on annotations from Gencode version 26
Expression values were also computed using RSEM version 1.2.28 based on Gencode version 26 annotations (based on Ensembl 88)
genome build: GRCh38
processed data files format and content: Readcounts: Tab-delimited file where columns are Ensembl gene identifier, counts for unstranded RNA, counts for 1st strand aligned, counts for 2nd strand aligned
processed data files format and content: RSEM: Tab-delimited file where columns correspond to a standard RSEM output (first line identifies the column)

source_name: Xenograft model bone marrow cells

extract_protocol:
KAPA RNA HyperPrep PolyA
TriZol + RNeasy mini Kit (Qiagen, #74106)
see SAMPLES section

tag: leukemia subtype

value: AML

tag: gene fusion

tag: cell sorting (facs or macs)


title: Adv-p62 wild type [W1]


title: CB-CD34+-4

source_name: Normal umbilical cord blood cells

extract_protocol:
KAPA RNA Stranded PolyA
TriZol + RNeasy mini Kit (Qiagen, #74106)
see SAMPLES section

value: CD34 positive


title: Adv-GFP [G2]


title: control 1 (non adenovirus infected) [N1]


title: FH4WTF

value: Wild type B6 mice


title: FH7NRFM


title: CB-CD34+-3


title: FH6NRFM


title: xB-ALL-2_D918

source_name: Xenograft model spleen cells

value: B-ALL

value: CD19 positive


title: FH8NRFM

source_name: liver


title: FH3WTF


title: FH5WTF


title: Adv-GFP [G1]


title: FH1WTM


title: xB-ALL-4_E755


title: FH2WTM


title: xAML-2_F737


title: pAMKL-3P_BM_019

source_name: Patient bone marrow cells (progression)

value: AMKL


title: pAMKL-3D

source_name: Patient bone marrow cells (diagnostic)


title: xB-ALL-1_C384


title: xAMKL-5_E890

value: GFP positive


title: xAMKL-1_C362_CD3

data_processing:
Basecalls performed using Illumina RTA 1.18.64
Samples demultiplexed using bcl2fastq 2.17
Sequenced reads were trimmed for sequencing adapters and low quality extrimities and mapped to the reference genome using STAR version 2.5.1b
Readcounts were computed by STAR during sequence alignment based on annotations from Gencode version 26
Expression values were also computed using RSEM version 1.2.28 based on Gencode version 26 annotations (based on Ensembl 88)
Genome_build: GRCh38
Supplementary_files_format_and_content: Readcounts: Tab-delimited file where columns are Ensembl gene identifier, counts for unstranded RNA, counts for 1st strand aligned, counts for 2nd strand aligned
Supplementary_files_format_and_content: RSEM: Tab-delimited file where columns correspond to a standard RSEM output (first line identifies the column)

extract_protocol:
KAPA RNA HyperPrep PolyA
TriZol + RNeasy mini Kit (Qiagen, #74106)

value: AMKL-associated

value: CD3 positive


title: pAML-6_BM_005


title: xAML-1_E385


title: CB-CD34+-2


title: xAMKL-3_E745

value: GFP positive, CD3 negative


title: xAMKL-1_C362


title: xB-ALL-3_D917


title: xAMKL-2_D922


title: pAMKL-5_BM_007


title: pAMKL-2_14H014


title: pAMKL-1_12H093


title: pAMKL-4_15H014


title: CD4 Zbtb7b KO Thpok-RV Replicate 2

status: Public on Aug 15 2019

data_processing:
Illumina RTA 1.18.61 software used for basecalling.
Samples demultiplexed using Bcl2fastq 2 software
Raw RNAseq fastq reads were trimmed with Trimmomatic and aligned to mouse genome using STAR
Gene-assignment and count of RNA reads were performed with Htseq
Genome_build: mm10
Supplementary_files_format_and_content: FPM normalization was performed with the FPM function of the DEseq2 package

source_name: Spleen

extract_protocol:
RNA was isolated using the RNeasy Plus Micro kit (Qiagen)
SMARTer Ultra Low Input reagent (Takara) and Nextera XT DNA (Illumina)
Libraries were pooled and  sequenced with paired-end reads of 126bp on a HiSeq2500 sequencer (Illumina) to reach 50 million read pairs per sample

value: Spleen

tag: infection

value: LCMV Arm d7 pi

tag: population

value: SMARTA

tag: genotype

value: Ox40-Cre+ Zbtb7bfl/fl


title: CD4 Zbtb7b KO Thpok-RV Replicate 3

tag: tissue


title: CD4 Zbtb7b KO Thpok-RV Replicate 1


title: CD4 Zbtb7b KO Bcl6-RV Replicate 3


title: CD4 Zbtb7b KO Bcl6-RV Replicate 2


title: CD4 Zbtb7b KO Bcl6-RV Replicate 1


title: CD4 Zbtb7b KO Empty-RV Replicate 3


title: CB-CD34+-1


title: CD4 Zbtb7b KO Empty-RV Replicate 2


title: CD4 WT Empty-RV Replicate 2


title: CD4 Zbtb7b KO Empty-RV Replicate 1


title: CD4 WT Empty-RV Replicate 3


title: CD4 WT Empty-RV Replicate 1


title: Sample 14: CD8 Ctrl Replicate 1

value: Ox40-Cre+ Zbtb7bwt/wt


title: Sample 13: CD8 Ctrl Replicate 1


title: Sample 9: CD4 CXCR5 Zbtb7b KO Replicate 3

value: GP66:I-Ab+ CXCR5int


title: Sample 10: CD4 Tfh Ctrl Replicate 1

value: GP66:I-Ab+ CXCR5high PD1+


title: Sample 11: CD4 Tfh Ctrl Replicate 2


title: Sample 7: CD4 CXCR5 Zbtb7b KO Replicate 1


title: Sample 4: CD4 CXCR5 Ctrl Replicate 1


title: Sample 12: CD4 Tfh Ctrl Replicate 3


title: Sample 5: CD4 CXCR5 Ctrl Replicate 2


title: Sample 6: CD4 CXCR5 Ctrl Replicate 3


title: Sample 1: CD4 Th1 Ctrl Replicate 1

value: GP66:I-Ab+ CXCR5-


title: Sample 3: CD4 Th1 Ctrl Replicate 3


title: Sample 2: CD4 Th1 Ctrl Replicate 2


title: Sample 15: CD8 Ctrl Replicate 1


title: PCa_ TRAMP+_3

status: Public on Jun 26 2019

description:
TRAMP+;p62f/f (wild type)
Processed data file RNASeq prostate tumor

data_processing:
FASTq files were processed using RNAseq Alignment module in BaseSpace (Illumina), using default parameters. Reads were aligned to mm10 genome using STAR. Differentially expressed genes were calculated using DeSeq2.
Genome_build: mm10
Supplementary_files_format_and_content: processed data file consist of an excel file matrix with gene symbols and FPKM counts for all samples

source_name: Mouse TRAMP-driven prostate tumor

extract_protocol:
Tissues were removed, flash frozen on dry ice, and RNA was harvested using Trizol reagent and purified using Omnigen RNA extraction kit. PolyA RNA was isolated using the NEBNext® Poly(A) mRNA Magnetic Isolation Module and barcoded libraries were made using the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina®(NEB, Ipswich MA).  Libraries were pooled and single end sequenced (1X75) on the Illumina NextSeq 500 using the High output V2 kit (Illumina Inc., San Diego CA).
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Regular diet

tag: strain

value: Prostate

tag: age

value: 30-35 weeks-old

value: PCa_ TRAMP+ (adipocyte p62 wild type)


title: AT_ TRAMP+_1

description:
TRAMP+;p62f/f (wild type)
Processed data file RNASeq adipose tissue

source_name: Mouse epidydimal adipose tissue

value: Epidydimal adipose tissue

value: AT_ TRAMP+ (adipocyte p62 wild type)


title: PCa_ TRAMP+_2


title: Sample 8: CD4 CXCR5 Zbtb7b KO Replicate 2


title: AT_ TRAMP+/p62Adipo_1

description:
TRAMP+;p62aP2KO (p62KO in adipocytes)
Processed data file RNASeq adipose tissue

value: AT_ TRAMP+/p62Adipo (adipocyte p62 KO)


title: AT_ TRAMP+_2


title: PCa_ TRAMP+/p62Adipo_3

description:
TRAMP+;p62aP2KO (p62KO in adipocytes)
Processed data file RNASeq prostate tumor

value: PCa_ TRAMP+/p62Adipo (adipocyte p62 KO)


title: AT_ TRAMP+/p62Adipo_2


title: PCa_ TRAMP+_4


title: AT_ TRAMP+_3


title: PCa_ TRAMP+/p62Adipo_1


title: PCa_ TRAMP+_1


title: AT_ TRAMP+/p62Adipo_3


title: Sham-Night Noise Sham-ADX sample c

status: Public on Jun 15 2019

description: ShNNShADXc

data_processing:
Reads were mapped with TopHat2 v.2.0.11.
Reads per gene feature were counted with HTSeq v.0.6p1.
Genome_build: mm10
Supplementary_files_format_and_content: raw counts

source_name: Sham-Night Noise Sham-ADX_cochlea total RNA

extract_protocol:
Cochlear RNAs were extracted as previously described (Patil et al, 2014) with classical methods using guanidium thiocyanate lysis buffer followed by purification on the ZymoResearch columns following the manufacturer's protocol
Total RNA was quantified with a Nanodrop and RNA integrity assessed with a Bioanalyzer (Agilent Technologies). The TruSeq mRNA stranded kit from Illumina was used for the library preparation with 300 ng of total RNA as input. Library molarity and quality was assessed with the Qubit and Tapestation using a DNA High sensitivity chip (Agilent Technologies). Pools of 6 libraries were loaded at 8.5 pM for clustering on a Single-read Illumina Flow cell. Reads of 50 bases were generated using the TruSeq SBS HS v3 chemistry on an Illumina HiSeq 2500 sequencer.

value: CBA/Sca

value: Cochlea

value: 12 weeks

tag: circadian time

tag: status

value: Sham-operated

value: Sham Night Noise


title: Sham-Night Noise Sham-ADX sample b

description: ShNNShADXb


title: Sham-Night Noise ADX sample c

description: ShNNADXc

source_name: Sham-Night Noise ADX_cochlea total RNA

value: adrenalectomized


title: Sham-Night Noise ADX sample b

description: ShNNADXb


title: Sham-Night Noise Sham-ADX sample a

description: ShNNShADXa


title: Sham-Night Noise ADX sample a

description: ShNNADXa


title: Sham-Day Noise ADX sample a

description: ShDNADXa

source_name: Sham-Day Noise ADX_cochlea total RNA

value: Sham Day Noise


title: Sham-Day Noise ADX sample c

description: ShDNADXc


title: Sham-Day Noise Sham-ADX sample b

description: ShDNShADXb

source_name: Sham-Day Noise Sham-ADX_cochlea total RNA


title: Sham-Day Noise Sham-ADX sample c

description: ShDNShADXc


title: Sham-Day Noise Sham-ADX sample a

description: ShDNShADXa


title: PCa_ TRAMP+/p62Adipo_2


title: Sham-Day Noise ADX sample b

description: ShDNADXb


title: Night Noise Sham-ADX sample c

description: NNShADXc

source_name: Night Noise Sham-ADX_cochlea total RNA

value: Night Noise


title: Day Noise ADX sample c

description: DNADXc

source_name: Day Noise ADX_cochlea total RNA

value: Day Noise


title: Night Noise ADX sample b

description: NNADXb

source_name: Night Noise ADX_cochlea total RNA


title: Night Noise Sham-ADX sample a

description: NNShADXa


title: Night Noise ADX sample a

description: NNADXa


title: Day Noise Sham-ADX sample b

description: DNShADXb

source_name: Day Noise Sham-ADX_cochlea total RNA


title: Night Noise ADX sample c

description: NNADXc


title: Day Noise ADX sample a

description: DNADXa


title: Day Noise ADX sample b

description: DNADXb


title: Day Noise Sham-ADX sample a

description: DNShADXa


title: Day Noise Sham-ADX sample c

description: DNShADXc


title: xAMKL-2_D922_CD3

source_name: Xenograft model bone marrow cells

value: AMKL-associated

value: CD3 positive


title: Night Noise Sham-ADX sample b

description: NNShADXb


title: sha_CT12.3

data_processing:
Sequences were aligned to the full reference genome index build with Ensembl annotation to improve accuracy on splices junctions using STAR 2.4.0i
A custom Perl script was used to quantify uniquely mapped reads. For each Ensembl gene, all exons of the respective annotated protein-coding transcripts were considered.
Genome_build: mm10
Supplementary_files_format_and_content: raw counts

source_name: Cochlea Total RNA

extract_protocol:
Cochlear RNAs were extracted as previously described (Patil et al, 2014) with classical methods using guanidium thiocyanate lysis buffer followed by purification on the miRNeasy Mini columns (Qiagen) following the manufacturer's protocol
Total RNA was quantified with a Nanodrop and RNA integrity assessed with a Bioanalyzer (Agilent Technologies). The TruSeq mRNA stranded kit from Illumina was used for the library preparation with 300 ng of total RNA as input. Library molarity and quality was assessed with the Qubit and Tapestation using a DNA High sensitivity chip (Agilent Technologies). Pools of 6 libraries were loaded at 8.5 pM for clustering on a Single-read Illumina Flow cell. Reads of 100 bases were generated using the TruSeq SBS HS v3 chemistry on an Illumina HiSeq 2500 sequencer.

value: sham-operated


title: sha_CT16.1


title: sha_CT24.3


title: sha_CT16.3


title: sha_CT24.1


title: sha_CT24.2


title: sha_CT20.1


title: sha_CT08.2


title: sha_CT12.1


title: sha_CT08.3


title: sha_CT16.2


title: sha_CT08.1


title: sha_CT12.2


title: sha_CT04.2


title: sha_CT04.1


title: sha_CT04.3


title: sha_CT20.3


title: adx_CT24.2


title: adx_CT20.2


title: adx_CT24.1


title: adx_CT24.3


title: adx_CT16.3


title: adx_CT20.3


title: adx_CT16.2


title: adx_CT16.1


title: sha_CT20.2


title: adx_CT12.3


title: adx_CT12.1


title: adx_CT04.3


title: adx_CT04.2


title: adx_CT08.2


title: adx_CT08.1


title: adx_CT04.1


title: MBL-mESC-IP-rep2 (dataset 4)

status: Public on Apr 26 2019

description: processed data file: mESC_HighFC.txt

data_processing:
Peak calling is adopted in the analysis of MeRIP data. Adaptors and low-quality reads (length shorter than 15) were trimmed from raw sequencing reads using cutadapt (https://cutadapt.readthedocs.io/en/stable/#).
Reads were then aligned to the relevant genome (human-hg19, mouse-mm10) using Tophat2 (version 2.2.1) without any gaps and allowed for at most two mismatches.
Peaks enriched in immunoprecipitation over input experiments were identified using exomePeak (Meng et al., 2013) and MeTPeak (Cui et al., 2016). For each cell line, only peaks identified (FDR≤0.05) in both replicates were considered as a candidate target. The further annotation of peaks was made use of PAVIS (https://manticore.niehs.nih.gov/pavis2/) and homer software (http://homer.ucsd.edu/homer/ngs/rnaseq/index.html).
All peaks listed in the .xls are with enrichment fold change (FC) larger than 3. These peaks appear consistently in both replicates.
Genome_build: hg19, mm10

source_name: MBL-mESC-IP

growth_protocol: Human HeLa, HepG2, and HEK293T cell lines and mouse embryonic stem cells (mESC) and mouse embryonic fibroblasts (MEF) used in this study were all purchased from ATCC. HeLa cell line was grown in DMEM (Gibco, 11965) media supplemented with 10% FBS and 1% 100X Pen/Strep (Gibco), while HepG2, HEK293T and MEF cell lines were maintained in DMEM (Gibco, 11995), supplemented with 10% FBS and 1% 100X Pen/Strep (Gibco). mESC cells were grown under a typical feeder-free culture condition: cells were cultured in DMEM (Gibco, 11995), supplemented with 15% FBS, 1% Pen/Strep (Gibco), 1X Glutamax (Gibco), 1X non-essential amino acids (Gibco), 1X 2-Mercaptoethanol (Gibco), and 1000 U/ml leukemia inhibitory factor (Millipore, ESG1107), together with two inhibitors: 3 uM CHIR99021 (Stemgent, dissolved in DMSO) and 1 uM PD0325901 (Stemgent, dissolved in DMSO). mESC cells were distinctively cultured on gelatinized culture plates (0.2% Gelatin). The culture of mESC cells were passaged every 2 days. All cells were cultured at 37 °C under 5.0% CO2.

extract_protocol:
2.0 ug poly(A)+ RNA was decapped and then fragmented into 50-100 nt using RNA fragmentation reagents (Thermo Fisher Scientific) following the manufacturer’s protocol. The fragmented poly(A)-tailed RNA was concentrated with RNA Clean & Concentrator (Zymo Research). The end structures of these RNA fragments were repaired by T4 PNK. The repaired RNA was then incubated with 2 ug MBL anti-m7G antibody (NEB anti-m6A antibody used for negative control in one cell line) in 200 ul IPP buffer (10mM Tris-Cl, pH=7.4; 150 mM NaCl; 0.1% NP-40) with freshly added 5% SUPERase-In RNase inhibitor (Thermo Fisher Scientific) at 4 °C for 2 h. 20 ul Dynabeads Protein G resins (Thermo Fisher Scientific) were washed twice, resuspended in 200 ul IPP buffer and incubated with the antibody-RNA mixture for another 2 h at 4 °C. The resins were then washed with IPP buffer at 4 °C for three times. RNA was finally eluted with proteinase K digestion. 90 ul 1X proteinase K digestion buffer (10X recipe: 10% SDS, 50 mM EDTA, 10 mM Tris-Cl (pH=7.4)) with 10 ul proteinase K was used to resuspend the resins, and the solution was incubated at 55 °C for 30 min. The second round of the digestion was prepared in the same way and incubated at 55 °C for another 30 min. Both flow-throughs were combined, and m7G-containing RNA was recovered with RNA Clean & Concentrator (Zymo Research).
Decapped and fragmented poly(A)+ RNA (as “input”) and immunoprecipitated RNA (as “IP”) were subjected to small RNA library construction following manufacturer’s instructions of NEBNext Small RNA Library Prep Set for Illumina (NEB). All libraries were sequenced on Illumina NextSeq 500 with single-end 80 bp read length.

treatment_protocol: For the libraries with siRNA knockdown of METTL1: AllStars negative control siRNA (Qiagen, SI03650318) was used as control siRNA in knockdown experiments. METTL1 siRNAs were ordered from Qiagen (siMETTL1-1: SI00076118). Transfection was performed with Lipofectamine RNAi-MAX (Invitrogen) following the manufacturer’s protocols. Generally, 15-cm plates of HeLa cells were prepared for 48 h knockdown. For the libraries with shRNA knockdown: the METTL1 shRNA vector was designed and constructed base on GPP with the construct IP as shRNA TRCN0000304184 and the target sequence as GGTGTATACCATAACCGATGT. Lentiviruses containing the METTL1 shRNAs were then produced in HEK293T cells with packaging plasmids. After 48 h, virus was were collected and used to infect HepG2 cells. Stable METTL1 knockdown cell lines were selected through 5 days of puromycin selection (2 μg/ml).

value: mouse embryonic stem cells (mESC)


title: MBL-MEF-IP-rep1 (dataset 4)

description: processed data file: MEF_HighFC.txt

source_name: MBL-MEF-IP

value: mouse embryonic fibroblasts (MEF)


title: MBL-MEF-IP-rep2 (dataset 4)

description: processed data file: MEF_HighFC.txt


title: MBL-mESC-input-rep1 (dataset 4)

description: processed data file: mESC_HighFC.txt

source_name: MBL-mESC-input


title: MBL-mECS-IP-rep1 (dataset 4)

description: processed data file: mESC_HighFC.txt

source_name: MBL-mECS-IP


title: MBL-mESC-input-rep2 (dataset 4)

description: processed data file: mESC_HighFC.txt


title: adx_CT20.1


title: MBL-MEF-input-rep1 (dataset 4)

description: processed data file: MEF_HighFC.txt

source_name: MBL-MEF-input


title: MBL-HepG2_shMETTL1-IP-rep2 (dataset 4)

description: processed data file: HepG2_shMETTL1_ConsistentDiff_peak.txt

value: HepG2 cells


title: MBL-HepG2_shMETTL1-input-rep2 (dataset 4)


title: MBL-HepG2_shMETTL1-IP-rep1 (dataset 4)


title: MBL-HepG2_shControl-input-rep1 (dataset 4)


title: MBL-HepG2_shControl-IP-rep2 (dataset 4)


title: MBL-HepG2_shControl-input-rep2 (dataset 4)


title: adx_CT12.2


title: MBL-HeLa_siControl-IP-rep2 (dataset 4)

description: processed data file: HeLa_siMETTL1_ConsistentDiff_peak.txt

value: HeLa cells


title: adx_CT08.3


title: MBL-HepG2_shControl-IP-rep1 (dataset 4)


title: MBL-HeLa_siMETTL1-input-rep1 (dataset 4)


title: MBL-HeLa_siMETTL1-IP-rep1 (dataset 4)


title: MBL-HeLa_siMETTL1-IP-rep2 (dataset 4)


title: MBL-MEF-input-rep2 (dataset 4)

description: processed data file: MEF_HighFC.txt


title: MBL-HeLa_siControl-IP-rep1 (dataset 4)


title: MBL-HeLa_siControl-input-rep2 (dataset 4)


title: MBL-HeLa_siControl-input-rep1 (dataset 4)


title: MBL-HepG2_shMETTL1-input-rep1 (dataset 4)


title: MBL-HEK293T-IP-rep1 (dataset 4)

description: processed data file: HEK293T_HighFC.txt

value: HEK293T cells


title: MBL-HEK293T-IP-rep2 (dataset 4)

description: processed data file: HEK293T_HighFC.txt


title: MBL-HeLa_siMETTL1-input-rep2 (dataset 4)


title: MBL-HEK293T-input-rep1 (dataset 4)

description: processed data file: HEK293T_HighFC.txt


title: MBL-HepG2-IP-rep1 (dataset 4)

description: processed data file: HepG2_HighFC.txt


title: MBL-HepG2-IP-rep2 (dataset 4)

description: processed data file: HepG2_HighFC.txt


title: MBL-HepG2-input-rep1 (dataset 4)

description: processed data file: HepG2_HighFC.txt


title: NEB-HeLa_m6A-IP-rep2 (dataset 4)

description: processed data file: HeLa_HighFC_m6A.txt


title: MBL-HeLa-IP-rep2 (dataset 4)

description: processed data file: HeLa_HighFC.txt

source_name: MBL-HeLa-IP


title: NEB-HeLa_m6A-IP-rep1 (dataset 4)


title: MBL-HeLa-input-rep2 (dataset 4)

description: processed data file: HeLa_HighFC.txt

source_name: MBL-HeLa-input


title: MBL-HeLa-IP-rep1 (dataset 4)

description: processed data file: HeLa_HighFC.txt


title: MBL-HepG2-input-rep2 (dataset 4)

description: processed data file: HepG2_HighFC.txt


title: MBL-HeLa-input-rep1 (dataset 4)

description: processed data file: HeLa_HighFC.txt