title: Ctrl_117_RBG19076

description: Ctrl_ICGScluster6_005_HSCs (CD31+kit+CD45-)


title: Treat_157_RBG19232

status: Public on Feb 03 2020

type: SRA

city: Shanghai

first: Xiaonan

last: Wang

address: 227 Chongqing South Road

department: School of Public Health

country: China

institute: Shanghai Jiao Tong University School of Medicine

description: Treated_ICGScluster7_006_HSCs (CD31+kit+CD45-)

library_source: transcriptomic

data_processing:
RNA-seq reads were aligned to mm10 using GSNAP (version 2015-09-29) with parameters (-B 5 -t 24 -n 1 -Q -N 1).
Reads in features were counted with htseq-count (HTSeq version 0.6.0) with the parameter (-s no).
Genome_build: mm10
Supplementary_files_format_and_content: HT-Seq counts / gene counts (.txt)

molecule: total RNA

organism: Mus musculus

source_name: the Aorta surrounded by Gonads and Mesonephros tissues (AGM)

extract_protocol:
CD31+kit+CD45- cells were FACS-purified.
Cells were sorted in 2.3 µl of lysis buffer containing 0.2% Triton X-100 (Sigma-Aldrich) and 1U of Superase-In RNase Inhibitor (Ambion) and processed following the Smart-Seq2 protocol.
Following preamplification, all samples were purified using Ampure XP beads (Beckman Coulter) at a ratio of 1:0.6. The cDNA was then quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) and size distributions were checked on high-sensitivity DNA chips (Agilent Bioanalyzer).
Libraries were prepared using the Illumina Nextera XT DNA preparation kit.
Single-cell RNA-seq analysis was performed as described previously (Picelli et al., 2014).
Pooled libraries were run on the Illumina Hi-Seq 4000.
Samples were used to construct Nextera XT libraries (Illumina) from 125 pg of preamplified cDNA. Libraries were purified and size selected (0.5X-0.7X) using Ampure XP beads. Then, libraries were quantified using KAPA qPCR quantification kit (KAPA Biosystems).

tag: cell type

value: Hematopoietic stem cells (CD31+kit+CD45-)

tag: treatment

tag: icgs cluster


title: Ctrl_113_RBG19028

description: Ctrl_ICGScluster2_041_HSCs (CD31+kit+CD45-)

value: Ctrl


title: Treat_169_RBG19281

description: Treated_ICGScluster1_066_HSCs (CD31+kit+CD45-)


title: Ctrl_115_RBG19052

description: Ctrl_ICGScluster1_053_HSCs (CD31+kit+CD45-)


title: Treat_152_RBG19267

description: Treated_ICGScluster5_003_HSCs (CD31+kit+CD45-)


title: Treat_154_RBG19291

description: Treated_ICGScluster4_006_HSCs (CD31+kit+CD45-)


title: Treat_150_RBG19243

description: Treated_ICGScluster1_062_HSCs (CD31+kit+CD45-)


title: Treat_153_RBG19279

description: Treated_ICGScluster1_063_HSCs (CD31+kit+CD45-)


title: STAG2nullB_Rep3

city: Dunedin

first: Jisha

last: Antony

state: Otago

address: 58 Hanover Street

department: Pathology

country: New Zealand

institute: University of Otago

description: CRISPR-Cas9 edited

data_processing:
Reads were adaptor and quality trimmed (Q20) using fastq-mcf.version v1.5.3.
Reads were aligned to the human genome GRCh37 (hg19) version using HISAT2 version 2.0.5 with gene annotation from Ensembl version 75.
Read counts were retrieved by exon and summarized by gene using featureCount version v1.5.3 and mapQ >10.
Genome_build: Human GRCh37 (hg19)
Supplementary_files_format_and_content: tab-delimited text file  contains gene read counts for each sample

organism: Homo sapiens

source_name: K562 cell line

growth_protocol: K562 were obtained from ATCC and grown in Isocove’s Modified Dulbecco’s Media (IMDM) (Life Technologies) containing 10% fetal bovine serum. Adherent STAG2 R614* mutant homozyogus cells were detached for subculture using trypsin-EDTA at 0.005% final concentration (Life technologies).

extract_protocol:
Total RNA was extracted using the NucleoSpin RNA kit (MACHEREY-NAGEL)
mRNA libraries were prepared using the NEBNext® Ultra™ RNA Library Prep Kit (Illumina®)

tag: cell line

tag: mutation

value: STAG2 R614*

tag: genotype


title: Treat_149_RBG19231

description: Treated_ICGScluster1_061_HSCs (CD31+kit+CD45-)


title: Treat_148_RBG19219

description: Treated_ICGScluster2_047_HSCs (CD31+kit+CD45-)


title: Treat_147_RBG19111

description: Treated_ICGScluster3_017_HSCs (CD31+kit+CD45-)


title: STAG2nullB_Rep2

description: CRISPR-Cas9 edited


title: Treat_146_RBG19099

description: Treated_ICGScluster2_046_HSCs (CD31+kit+CD45-)


title: Treat_145_RBG19087

description: Treated_ICGScluster1_060_HSCs (CD31+kit+CD45-)


title: STAG2nullA_Rep3

description: CRISPR-Cas9 edited


title: STAG2nullB_Rep1

description: CRISPR-Cas9 edited


title: Treat_144_RBG19075

description: Treated_ICGScluster1_059_HSCs (CD31+kit+CD45-)


title: STAG2nullA_Rep2

description: CRISPR-Cas9 edited


title: Ctrl_116_RBG19064

description: Ctrl_ICGScluster2_042_HSCs (CD31+kit+CD45-)


title: STAG2nullA_Rep1

description: CRISPR-Cas9 edited


title: Ctrl_114_RBG19040

description: Ctrl_ICGScluster1_052_HSCs (CD31+kit+CD45-)


title: STAG2heterozygous2_Rep2

description: CRISPR-Cas9 edited


title: Treat_151_RBG19255

description: Treated_ICGScluster3_018_HSCs (CD31+kit+CD45-)


title: STAG2heterozygous1_Rep3

description: CRISPR-Cas9 edited


title: Treat_143_RBG19063

description: Treated_ICGScluster1_058_HSCs (CD31+kit+CD45-)


title: Treat_141_RBG19039

description: Treated_ICGScluster1_057_HSCs (CD31+kit+CD45-)


title: Treat_142_RBG19051

description: Treated_ICGScluster6_012_HSCs (CD31+kit+CD45-)


title: Treat_140_RBG19027

description: Treated_ICGScluster2_045_HSCs (CD31+kit+CD45-)


title: Treat_139_RBG19302

description: Treated_ICGScluster2_044_HSCs (CD31+kit+CD45-)


title: Treat_138_RBG19290

description: Treated_ICGScluster1_056_HSCs (CD31+kit+CD45-)


title: K562WT_Rep3

description: original K562 cells

value: parental no mutation in STAG2

value: wild type


title: STAG2heterozygous1_Rep1

description: CRISPR-Cas9 edited


title: K562WT_Rep2

description: original K562 cells


title: STAG2heterozygous1_Rep2

description: CRISPR-Cas9 edited


title: Treat_136_RBG19266

description: Treated_ICGScluster3_015_HSCs (CD31+kit+CD45-)


title: Treat_137_RBG19278

description: Treated_ICGScluster3_016_HSCs (CD31+kit+CD45-)


title: Treat_135_RBG19254

description: Treated_ICGScluster1_055_HSCs (CD31+kit+CD45-)


title: K562WT_Rep1

description: original K562 cells


title: Treat_132_RBG19218

description: Treated_ICGScluster2_041_HSCs (CD31+kit+CD45-)


title: STAG2heterozygous2_Rep3

description: CRISPR-Cas9 edited


title: Treat_133_RBG19230

description: Treated_ICGScluster2_042_HSCs (CD31+kit+CD45-)


title: Treat_131_RBG19110

description: Treated_ICGScluster2_040_HSCs (CD31+kit+CD45-)


title: STAG2heterozygous2_Rep1

description: CRISPR-Cas9 edited


title: TS98_H3K27ac_BB497_T2

city: Manchester

first: Tim

middle: C

last: Somervaille

address: Wilmslow Rd

department: Manchester Institute

country: United Kingdom

institute: Cancer Research UK

library_source: genomic

data_processing:
Libraries were prepared for sequencing using a Microplex Library Preparation Kit (Diagenode). 200-800bp fragments were selected using AMPure beads (Beckman Coulter) and quantified by qPCR with a KAPA Library Quantification Kit (Kapa Biosystems). Sequencing was performed using a NextSeq desktop sequencing system (Illumina) with 75bp, paired-end high output generating 40-65M reads per sample. Reads were aligned to the human genome (hg38) using BWA-MEM v0.7.15 (http://bio-bwa.sourceforge.net/). Read duplicates were removed using Picard v2.1.0. Reads were further filtered using Bedtools v2.25.0 to keep only paired reads mapped to standard chromosomes and to remove reads with a mapping quality of less than 10. Reads mapped to blacklisted regions defined by ENCODE were then removed using Bedtools (http://mitra.stanford.edu/kundaje).
Genome_build: GRCh38
Supplementary_files_format_and_content: bigwig

molecule: genomic DNA

source_name: H3K27ac BB497 T2

growth_protocol:
Cyropreserved primary cells were thawed and immediately crosslinked for ChIP.
K562 and derived cell lines were cultured in RPMI 1640 medium (Sigma Aldrich) supplemented with 2mM L-Glutamine (Life Technologies) and 10% fetal bovine serum (Sigma Aldrich).

extract_protocol:
ChIP was performed using anti-H3K27ac (ab4729, Abcam) and anti-H3K9me3 (ab8898, Abcam). 10^8 cells were used for each precipitation as described by Lee et al. (2006). Cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature before the reaction was quenched with 0.125M glycine. Cell pellets were washed twice with PBS and nuclear lysates sonicated for 6 cycles using a Bioruptor® Pico (Diagenode). 10μg of antibody bound to 100μl of magnetic beads (Dynabeads Protein G, Invitrogen, Carlsbad, CA) was added to each sample and immunoprecipiation performed overnight on a rotator at 4°C and 20 rpm. After 5 washes with RIPA buffer (50mM HEPES pH 7.6, 1mM EDTA, 0.7% Na deoxycholate, 1% NP-40, 0.5M LiCl), chromatin IP-bound fractions were extracted by incubating for 15 min at 65°C with elution buffer (50mM TrisHCl pH8, 10mM EDTA, 1% SDS). Crosslinking was then reversed by incubation at 65°C for 6 hours. RNaseA (1mg/ml) and proteinase K (20mg/ml) were added to eliminate RNA and protein from the samples. DNA was extracted using phenol:chloroform:isoamyl alcohol and precipitated by adding 2 volumes of ice-cold 100% ethanol, glycogen (20μg/μl), 200mM NaCl and freezing at  -80°C for at least 1 hour. Pellets were washed with 70% ethanol and eluted in 50μl 10mM TrisHCl pH8.0.
Libraries were prepared for sequencing using a Microplex Library Preparation Kit (Diagenode). 200-800bp fragments were selected using AMPure beads (Beckman Coulter) and quantified by qPCR with a KAPA Library Quantification Kit (Kapa Biosystems).

value: Primary AML

tag: chip antibody

tag: chip antibody vendor

value: Abcam, Cambridge, UK

tag: chip antibody cat. #

tag: chip antibody lot#


title: TS98_H3K27ac_BB727_T1

source_name: H3K27ac BB727 T1


title: Treat_130_RBG19098

description: Treated_ICGScluster3_014_HSCs (CD31+kit+CD45-)


title: TS98_H3K27ac_BB54_T2

source_name: H3K27ac BB54 T2


title: Treat_129_RBG19086

description: Treated_ICGScluster1_054_HSCs (CD31+kit+CD45-)


title: Treat_128_RBG19074

description: Treated_ICGScluster1_053_HSCs (CD31+kit+CD45-)


title: TS98_H3K27ac_BB338_T2

source_name: H3K27ac BB338 T2


title: Treat_127_RBG19062

description: Treated_ICGScluster1_052_HSCs (CD31+kit+CD45-)


title: TS98_H3K27ac_BB171_T3

source_name: H3K27ac BB171 T3


title: TS98_H3K27ac_BB380_T1

source_name: H3K27ac BB380 T1


title: Treat_125_RBG19026

description: Treated_ICGScluster1_051_HSCs (CD31+kit+CD45-)


title: Treat_126_RBG19050

description: Treated_ICGScluster2_039_HSCs (CD31+kit+CD45-)


title: TS98_H3K27ac_BB14_T10

source_name: H3K27ac BB14 T10


title: Treat_124_RBG19301

description: Treated_ICGScluster1_050_HSCs (CD31+kit+CD45-)


title: TS98_H3K27ac_BB556_T2

source_name: H3K27ac BB556 T2


title: Treat_134_RBG19242

description: Treated_ICGScluster2_043_HSCs (CD31+kit+CD45-)


title: Treat_121_RBG19265

description: Treated_ICGScluster2_038_HSCs (CD31+kit+CD45-)


title: TS98_INPUT_BB556_T2

source_name: INPUT BB556 T2


title: Treat_123_RBG19289

description: Treated_ICGScluster1_049_HSCs (CD31+kit+CD45-)


title: TS93_H3K9me3_K562_R1_+dCas9_KRAB_+sgRNA_Promoter

source_name: H3K9me3 K562_R1 +dCas9_KRAB +sgRNA_Promoter

growth_protocol:
Cultured in RPMI 1640 medium (Sigma Aldrich) supplemented with 2mM L-Glutamine (Life Technologies) and 10% fetal bovine serum (Sigma Aldrich).
K562 and derived cell lines were cultured in RPMI 1640 medium (Sigma Aldrich) supplemented with 2mM L-Glutamine (Life Technologies) and 10% fetal bovine serum (Sigma Aldrich).


title: TS93_H3K9me3_K562_R1_+dCas9_KRAB_+sgRNA_E4

source_name: H3K9me3 K562_R1 +dCas9_KRAB +sgRNA_E4


title: Treat_120_RBG19253

description: Treated_ICGScluster1_047_HSCs (CD31+kit+CD45-)


title: TS93_H3K9me3_K562_R1_+dCas9_KRAB_+sgRNA_E3

source_name: H3K9me3 K562_R1 +dCas9_KRAB +sgRNA_E3


title: TS93_H3K9me3_K562_R1_+dCas9_KRAB_+sgRNA_E2

source_name: H3K9me3 K562_R1 +dCas9_KRAB +sgRNA_E2


title: TS93_H3K9me3_K562_R1_+dCas9_KRAB_+sgRNA_E1

source_name: H3K9me3 K562_R1 +dCas9_KRAB +sgRNA_E1


title: Treat_119_RBG19241

description: Treated_ICGScluster1_046_HSCs (CD31+kit+CD45-)


title: Treat_118_RBG19229

description: Treated_ICGScluster4_005_HSCs (CD31+kit+CD45-)


title: TS98_H3K27ac_BB225_T15


title: Treat_116_RBG19109

description: Treated_ICGScluster6_011_HSCs (CD31+kit+CD45-)


title: TS93_INPUT_K562_R1_+dCas9_KRAB

source_name: INPUT K562_R1 +dCas9_KRAB


title: TS93_H3K9me3_K562_R1_+dCas9_KRAB_+sgRNA_EV

source_name: H3K9me3 K562_R1 +dCas9_KRAB +sgRNA_EV


title: Treat_115_RBG19097

description: Treated_ICGScluster1_045_HSCs (CD31+kit+CD45-)

source_name: the Aorta surrounded by Gonads and Mesonephros tissues (AGM)


title: TS98_H3K27ac_BB546_T6

source_name: H3K27ac BB546 T6


title: TS75_H3K27ac_K562_R2


title: Treat_114_RBG19085

description: Treated_ICGScluster6_010_HSCs (CD31+kit+CD45-)


title: TS75_H3K27ac_K562_R1


title: TS75_H3K27ac_K562_S3


title: Treat_111_RBG19049

description: Treated_ICGScluster2_035_HSCs (CD31+kit+CD45-)


title: Treat_113_RBG19073

description: Treated_ICGScluster1_044_HSCs (CD31+kit+CD45-)


title: Treat_122_RBG19277

description: Treated_ICGScluster1_048_HSCs (CD31+kit+CD45-)


title: Treat_112_RBG19061

description: Treated_ICGScluster2_036_HSCs (CD31+kit+CD45-)


title: Treat_110_RBG19037

description: Treated_ICGScluster6_009_HSCs (CD31+kit+CD45-)


title: Treat_109_RBG19025

description: Treated_ICGScluster3_013_HSCs (CD31+kit+CD45-)


title: TS75_H3K27ac_K562_S1


title: TS75_H3K27ac_K562_S2


title: TS75_INPUT_K562_S2


title: Ctrl_112_RBG19300

description: Ctrl_ICGScluster1_051_HSCs (CD31+kit+CD45-)


title: K562_R3_Rep2

data_processing:
Paired-end reads were mapped to the human genome (GRCh38 Gencode release 25) with STAR version 2.4.2a with the settings --outFilterMultimapNmax 20, --outFilterType BySJout,  --alignSJoverhangMin 8 and --quantMode GeneCounts using the matching feature annotation GTF file for GRCh38 Gencode release 25. Differential gene expression analysis was performed using DESeq2 version 1.20.0
Genome_build: GRCh38
Supplementary_files_format_and_content: tab-delimited text files include raw read count values per gene for each sample

growth_protocol: Cultured in RPMI 1640 medium (Sigma Aldrich) supplemented with 2mM L-Glutamine (Life Technologies) and 10% fetal bovine serum (Sigma Aldrich).

extract_protocol:
Total RNA was extracted from sensitive or resistant K562 cells using QIAshredder spin columns and an RNeasy Plus Micro Kit (Qiagen, Manchester, UK) and its quality confirmed using an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA).
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: sensitivity phenotype

value: Resistant


title: Ctrl_111_RBG19288

description: Ctrl_ICGScluster2_040_HSCs (CD31+kit+CD45-)


title: TS75_H3K27ac_K562_R3


title: Ctrl_110_RBG19276

description: Ctrl_ICGScluster1_050_HSCs (CD31+kit+CD45-)


title: K562_R3_Rep1


title: Treat_117_RBG19217

description: Treated_ICGScluster2_037_HSCs (CD31+kit+CD45-)


title: K562_S3

value: Sensitive


title: Ctrl_109_RBG19264

description: Ctrl_ICGScluster1_049_HSCs (CD31+kit+CD45-)


title: Ctrl_108_RBG19252

description: Ctrl_ICGScluster1_048_HSCs (CD31+kit+CD45-)


title: K562_R2_Rep1


title: K562_R2_Rep2


title: Ctrl_107_RBG19240

description: Ctrl_ICGScluster2_039_HSCs (CD31+kit+CD45-)


title: Ctrl_106_RBG19228

description: Ctrl_ICGScluster2_038_HSCs (CD31+kit+CD45-)


title: K562_R1_Rep2


title: K562_R1_Rep1


title: K562_S2


title: Treat_105_RBG19072

description: Treated_ICGScluster1_041_HSCs (CD31+kit+CD45-)


title: Treat_107_RBG19096

description: Treated_ICGScluster1_042_HSCs (CD31+kit+CD45-)


title: Treat_155_RBG19303

description: Treated_ICGScluster2_048_HSCs (CD31+kit+CD45-)


title: Treat_106_RBG19084

description: Treated_ICGScluster2_034_HSCs (CD31+kit+CD45-)


title: K562_S1


title: Treat_108_RBG19108

description: Treated_ICGScluster1_043_HSCs (CD31+kit+CD45-)


title: [ATAC-seq] MEF flagMyod1 (rep2)

city: Singapore

first: Qian Yi

last: Lee

address: Biopolis Drive

department: R&D

country: Singapore

institute: Nuevocor

data_processing:
Adaptor trimming with in-house algorithm
Aligning reads to mm10 using Bowtie 2.1.0 with following parameter:  --very-sensitive
Removed mitochondria reads with awk '$3!="chrM"'
Removed duplicate reads with picard-tools v1.79 using the following parameters: -Xmx4G VALIDATION_S  TRINGENCY=LENIENT ASSUME_SORTED=true REMOVE_DUPLICATES=true
RPKM values were calculated counting reads within ±100bp from merged FLAG ChIP peak summits
RPKM values were normalized using the normalize.quantiles function in the preprocessCore package in R
Genome_build: mm10
Supplementary_files_format_and_content: Processed tab-delimited file contains peak information (chr, start, stop) and normalized RPKM values for each replicate. Includes MEF only, MEF+rtTA and MEF+Ascl1 data from GSE101397.

source_name: Mouse embryonic fibroblasts

growth_protocol: Homozygous TauEGFP knock-in mice were bred with C57B/6 mice (Charles River) to generate heterozygous embryos. Heterozygous TauEGFP knock-in TauEGFP mouse embryonic fibroblasts (MEFs) were then isolated from E13.5 embryos.

extract_protocol:
At 48hr post-dox induction, ATAC-seq was performed as described in Buenrostro, et al., Nat. Methods (2016).
ATAC-seq libraries were sequenced on Illumina HiSeq 2000.

treatment_protocol: P3 MEFs were co-infected with reverse tetracycline transactivator (rtTA) and doxycycline (dox) inducible Myod1 overnight and then cultured in MEF media (DMEM; Invitrogen) containing 10% cosmic calf serum (CCS; Hyclone), beta-mercaptoethanol (Sigma), non-essential amino acids, sodium pyruvate and penicillin/streptomycin (all from Invitrogen) supplemented with 2 µg/ml dox (Sigma) for 48h.

value: Mouse embryonic fibroblasts

tag: transgenes

value: flagMyod1 + rtTA

tag: days post transgene induction

value: 2 days


title: [ChIP-seq, H3K27ac] MEF novirus input

description: AM_mergedpeaks_+-500bp_noAMcds_H3K27ac_rpkm.txt

data_processing:
Reads were aligned to the mm10 reference sequence using Bowtie 2.1.0 with an additional -5 10 parameter.
Reproducible IDR peaks from the Ascl1 and Myod1 FLAG-ChIP-seq dataset were merged using Diffbind 1.16.3 with minOverlap = 2.
The number of reads that fall within the 500bp region is counted for each peak, then normalized by the total number of reads per replicate and by the interval size (RPKM = count/total read counts/500 * 10^9).
The RPKM value is further normalized by the average signal per sample (norm. RPKM = RPKM/mean RPKM for each replicate).
Genome_build: mm10
Supplementary_files_format_and_content: Processed tab-delimited file contains peak information (chr, start, stop) and normalized RPKM values for each replicate.

extract_protocol:
Approximately 30 million cells were used for each experiment. Chromatin was sheared using the Covaris sonicator until DNA was fragmented to 200-500bp. For each reaction 1% of sonicated chromatin was kept as input DNA. 5ug of H3K27ac (Abcam ab4729) antibody was added to remaining chromatin and incubated overnight at 4°C. 100uL of protein G Dynabeads (Thermo Fisher; 10003D) were added to each ChIP reaction and incubated for at least 4h at 4°C. Beads were then washed, eluted and reverse cross-linked overnight. After reverse cross-linking, isolated DNA was RNase treated for 30min at 37°C, purified using QIAquick PCR purification columns (Qiagen) and eluted in DEPC water.
ChIP-seq libraries were prepared using the NEBNext library prep kit (NEB; E6240) following the supplier’s protocols with slight modifications. Adaptor ligation was performed using regular T4 ligase and ligase buffer. Size selection (200-500bp) was performing after adaptor ligation and PCR enrichment using 2% low-melt agarose gel (Lonza; 50080), and DNA was purified using the QiaQuick gel extraction columns from Qiagen.

treatment_protocol: P3 MEFs were co-infected with reverse tetracycline transactivator (rtTA) and doxycycline (dox) inducible Ascl1 or Myod1 overnight and then cultured in MEF media (DMEM; Invitrogen) containing 10% cosmic calf serum (CCS; Hyclone), beta-mercaptoethanol (Sigma), non-essential amino acids, sodium pyruvate and penicillin/streptomycin (all from Invitrogen) supplemented with 2 µg/ml dox (Sigma) for 48h.

value: no virus

value: input


title: [ChIP-seq, H3K27ac] MEF novirus IP (rep2)

value: H3K27ac (Abcam ab4729)


title: [ChIP-seq, H3K27ac] MEF novirus IP (rep1)


title: Treat_102_RBG19036

description: Treated_ICGScluster2_032_HSCs (CD31+kit+CD45-)


title: Treat_103_RBG19048

description: Treated_ICGScluster2_033_HSCs (CD31+kit+CD45-)


title: [ChIP-seq, H3K27ac] MEF rtTA IP (rep2)

value: rtTA


title: [ChIP-seq, H3K27ac] MEF rtTA input (rep2)


title: [ChIP-seq, H3K27ac] MEF rtTA IP (rep1)


title: Treat_101_RBG19024

description: Treated_ICGScluster1_039_HSCs (CD31+kit+CD45-)


title: Ctrl_103_RBG19287

description: Ctrl_ICGScluster2_035_HSCs (CD31+kit+CD45-)


title: [ChIP-seq, H3K27ac] MEF flagMyod1 input


title: Ctrl_104_RBG19299

description: Ctrl_ICGScluster2_036_HSCs (CD31+kit+CD45-)


title: [ChIP-seq, H3K27ac] MEF rtTA input (rep1)


title: Ctrl_105_RBG19216

description: Ctrl_ICGScluster2_037_HSCs (CD31+kit+CD45-)


title: Ctrl_102_RBG19275

description: Ctrl_ICGScluster1_047_HSCs (CD31+kit+CD45-)


title: Ctrl_101_RBG19263

description: Ctrl_ICGScluster2_034_HSCs (CD31+kit+CD45-)


title: [ChIP-seq, H3K27ac] MEF flagMyod1 IP (rep1)


title: [ChIP-seq, H3K27ac] MEF flagMyod1 IP (rep2)


title: Ctrl_100_RBG19239

description: Ctrl_ICGScluster1_046_HSCs (CD31+kit+CD45-)


title: Treat_104_RBG19060

description: Treated_ICGScluster1_040_HSCs (CD31+kit+CD45-)


title: Treat_100_RBG19107

description: Treated_ICGScluster2_031_HSCs (CD31+kit+CD45-)


title: [ChIP-seq, H3K27ac] MEF flagAscl1 input (rep1)

value: flagAscl1 + rtTA


title: [ChIP-seq, FLAG] MEF flagMyod1+Myt1l input (rep1)

description: TGM_flagMyod+Myt1l_combinedinput.idrpeaks.0.1.merged.bed

data_processing:
Reads were aligned to the mm10 reference sequence using Bowtie 2.1.0 with an additional -5 10 parameter.
Peak calling was performed using MACS 2.1.1 .
For experiments with two replicates, reproducible peaks were selected using IDR 2.0.2. with cutoff of idr ≤ 0.1 using the recommended pipeline for MACS2. For experiments with ≥ 3 replicates, pairwise IDR was performed and reproducible peaks (idr ≤ 0.1) for each pair were merged into a single peak list using Diffbind 1.16.3 with minOverlap = number of replicates.
Genome_build: mm10
Supplementary_files_format_and_content: Tab delimited file of merged peaks (chr, start, stop, name, p-value, strand)

extract_protocol:
FLAG-tagged TFs were immunoprecipitated using FLAG M2 antibody conjugated beads (Sigma F2426). Approximately 60-100 million cells were used per replicate. Beads (~100uL slurry per sample) were washed thrice with IP dilution buffer (1% Triton X-100, 2mM EDTA pH8.0, 20mM Tris-HCl pH8.0, 150mM NaCl, 1mM DTT, 100uM PMSF), blocked overnight in IP dilution buffer with 0.1% BSA and 0.06% sheared salmon sperm DNA (Thermo Fisher; AM9680), then washed again three times with IP dilution buffer before use. Nuclei were isolated by incubating with cell lysis buffer (5mM HEPES pH7.9, 85mM KCl, 0.5% NP40, 100uM PMSF, protease inhibitors from Roche) for 10min on ice and centrifuged at 5000rpm for 5min at 4°C, then lysed with nuclear lysis buffer (50mM Tris-HCl pH8.0, 10mM EDTA pH8.0, 1% SDS, 100uM PMSF, protease inhibitors) for 10min on ice. Chromatin was sheared using either the Bioruptor (Diagenode) or Covaris sonicator until DNA was fragmented to 200-500bp. Sheared chromatin was diluted using 3X volume of IP dilution buffer and pre-cleared for at least 4hr at 4°C using IgG beads (Sigma A0919). 1% of pre-cleared chromatin was kept as input, and the remainder was incubated with FLAG beads overnight at 4°C. Beads were washed 8 times with IP wash buffer (20mM Tris-HCl pH8.0, 2mM EDTA pH8.0, 250mM NaCl, 1% NP40, 0.05% SDS, 100uM PMSF) and once with TE buffer with 100uM PMSF. Beads and 1% input samples were reverse cross-linked overnight in IP elution buffer (50mM NaHCO3, 1% SDS) at 65°C. Isolated DNA was RNase treated for 30min at 37°C, purified using QIAquick PCR purification columns (Qiagen) and eluted in DEPC water.
ChIP-seq libraries were prepared using the NEBNext library prep kit (NEB; E6240) following the supplier’s protocols with slight modifications. Adaptor ligation was performed using regular T4 ligase and ligase buffer. Size selection (200-500bp) was performing after adaptor ligation and PCR enrichment using 2% low-melt agarose gel (Lonza; 50080), and DNA was purified using the QiaQuick gel extraction columns from Qiagen.

value: flagMyod1 + Myt1l + rtTA


title: Ctrl_099_RBG19227

description: Ctrl_ICGScluster6_004_HSCs (CD31+kit+CD45-)

value: Hematopoietic stem cells (CD31+kit+CD45-)

value: Ctrl


title: [ChIP-seq, H3K27ac] MEF flagAscl1 input (rep2)


title: [ChIP-seq, FLAG] MEF flagMyod1+Myt1l input (rep2)


title: [ChIP-seq, H3K27ac] MEF flagAscl1 IP (rep1)


title: Treat_097_RBG19071

description: Treated_ICGScluster2_030_HSCs (CD31+kit+CD45-)


title: Ctrl_098_RBG19215

description: Ctrl_ICGScluster2_033_HSCs (CD31+kit+CD45-)


title: Treat_096_RBG19059

description: Treated_ICGScluster6_008_HSCs (CD31+kit+CD45-)


title: Treat_094_RBG19035

description: Treated_ICGScluster1_035_HSCs (CD31+kit+CD45-)


title: Treat_095_RBG19047

description: Treated_ICGScluster1_036_HSCs (CD31+kit+CD45-)


title: [ChIP-seq, FLAG] MEF flagMyod1(Ascl1-Basic+C-term) input (rep2)

description:
Construct swapping out Myod1's basic and C-term domain with corresponding domains from Ascl1
TGM_flagMyod-A1-B-C_combinedinput.idrpeaks.0.1.merged.bed

value: flagMyod1(Ascl1-B+C) + rtTA


title: [ChIP-seq, FLAG] MEF flagMyod1(Ascl1-Basic+C-term) input (rep1)


title: [ChIP-seq, FLAG] MEF flagMyod1(Ascl1-Basic+C-term) IP (rep2)

value: FLAG-conjugated beads (Sigma F2426)


title: [ChIP-seq, FLAG] MEF flagMyod1(Ascl1-Basic+C-term) IP (rep1)


title: Ctrl_097_RBG19298

description: Ctrl_ICGScluster2_032_HSCs (CD31+kit+CD45-)


title: Treat_093_RBG19023

description: Treated_ICGScluster1_034_HSCs (CD31+kit+CD45-)


title: Ctrl_096_RBG19286

description: Ctrl_ICGScluster2_031_HSCs (CD31+kit+CD45-)


title: [ChIP-seq, H3K27ac] MEF flagAscl1 IP (rep2)

description: AM_mergedpeaks_+-500bp_noAMcds_H3K27ac_rpkm.txt


title: Treat_099_RBG19095

description: Treated_ICGScluster1_038_HSCs (CD31+kit+CD45-)


title: [ChIP-seq, FLAG] MEF rtTA-control input (rep2)

description: TGM_rtTA_Rallinput.idrpeaks.0.1.merged.bed


title: Ctrl_095_RBG19274

description: Ctrl_ICGScluster2_030_HSCs (CD31+kit+CD45-)


title: [ChIP-seq, FLAG] MEF rtTA-control IP (rep2)


title: Treat_098_RBG19083

description: Treated_ICGScluster1_037_HSCs (CD31+kit+CD45-)


title: Ctrl_094_RBG19250

description: Ctrl_ICGScluster1_045_HSCs (CD31+kit+CD45-)


title: [ChIP-seq, FLAG] MEF rtTA-control input (rep1)


title: Ctrl_092_RBG19226

description: Ctrl_ICGScluster2_029_HSCs (CD31+kit+CD45-)


title: [ChIP-seq, FLAG] MEF flagMyod1 input (rep4)

description: TGM_flagMyoD_MDallinput.idrpeaks.0.1.merged.bed


title: Ctrl_093_RBG19238

description: Ctrl_ICGScluster1_044_HSCs (CD31+kit+CD45-)


title: [ChIP-seq, FLAG] MEF rtTA-control IP (rep1)


title: [ChIP-seq, FLAG] MEF flagMyod1 input (rep3)


title: [ChIP-seq, FLAG] MEF flagMyod1+Myt1l IP (rep1)

description: TGM_flagMyod+Myt1l_combinedinput.idrpeaks.0.1.merged.bed


title: [ChIP-seq, FLAG] MEF flagMyod1+Myt1l IP (rep2)


title: Treat_092_RBG19106

description: Treated_ICGScluster4_004_HSCs (CD31+kit+CD45-)


title: [ChIP-seq, FLAG] MEF flagMyod1 IP (rep4)


title: Treat_090_RBG19070

description: Treated_ICGScluster1_032_HSCs (CD31+kit+CD45-)


title: Treat_091_RBG19094

description: Treated_ICGScluster1_033_HSCs (CD31+kit+CD45-)


title: [ChIP-seq, FLAG] MEF flagMyod1 IP (rep3)


title: Treat_089_RBG19058

description: Treated_ICGScluster6_007_HSCs (CD31+kit+CD45-)


title: Treat_088_RBG19046

description: Treated_ICGScluster2_029_HSCs (CD31+kit+CD45-)


title: [ChIP-seq, FLAG] MEF flagAscl1 input (rep3)

description: TGM_flagAscl1_A1allinput.idrpeaks.0.1.merged.bed


title: Kat2a-KO cells

city: Cambridge

first: Cristina

last: Pina

address: Long Road

department: Department of Haematology Cambridge Biomedical Campus NHS - Blood and Transplant

institute: University of Cambridge

description: barcodes.tsv, genes.tsv, matrix.mtx

data_processing:
The files are processed using Cellranger (v.1.3.1) software provided by 10X genomics platform.
The processed data files after processing will generate gene-cell matrix combined for both the genotypes. There will be in total 8309 cells, first 4240 cells are Kat2a-KO (barcode ending in suffix 1) genotype and next 4069 cells are of Kat2-WT (Barcode ending in suffix 2) genotype.
Genome_build: mm10
Supplementary_files_format_and_content: For Kat2a-WT and Kat2a-KO samples, 3 files are provided as processed data.

source_name: Bone marrow cells

growth_protocol: MLL-AF9 AMLs generated from lineage-depleted BM cells retrovirally (RV)-transduced for expression of the MLL-AF9 fusion oncogene and transplanted into lethally irradiated mouse recipients.

extract_protocol:
12K live cells per genotype pool were sorted on the basis of YFP expression (reporting MLL-AF9), Hoechst 32258 exclusion (live cells) and singlet configuration (pulse width) and used  for library preparation aiming at 6K single cells per sample.
10X Genomics single-cell RNA sequencing library preparation aiming at 6000 cells per genotype (2 samples)
10X Genomics single-cell RNA-seq and Illumina NextSeq-based sequencing

treatment_protocol: Kat2a Flox/Flox Mx1-Cre -/- (WT) or Mx1-Cre+/- (KO) mice were treated with pIpC for locus excision and BM collected 4-6 weeks later for RV transduction and injection into irradiated recipients. Terminal AML BM samples were collected (WT - 5; KO - 4) and the individual cell samples stored at -150C. Cells were thawed and pooled for library preparation.

value: Bone marrow cells

tag: disease state

value: MLL-AF9 leukemia


title: Kat2a-WT cells

description: barcodes.tsv, genes.tsv, matrix.mtx


title: Treat_087_RBG19034

description: Treated_ICGScluster1_031_HSCs (CD31+kit+CD45-)


title: Treat_085_RBG19213

description: Treated_ICGScluster3_011_HSCs (CD31+kit+CD45-)


title: Ctrl_091_RBG19214

description: Ctrl_ICGScluster1_043_HSCs (CD31+kit+CD45-)


title: Treat_084_RBG19201

description: Treated_ICGScluster1_030_HSCs (CD31+kit+CD45-)


title: Sample 21 EPF2p::AGO1DAH

city: Daejeon

first: Ji-Hwan

last: Park

address: 125 Gwahak-ro, Yuseong-gu

country: South Korea

institute: Korea Research Institute of Bioscience and Biotechnology (KRIBB)

description:
Transgenic plant harboring EPF2 promoter, GFP and AGO1DAH (slicer-defective AGO1) construct
Sample 21

data_processing:
After the library sequencing, the adapter sequences were trimmed, and the reads were filtered out with length < 15 bp.
The remained reads were then aligned to the Arabidopsis thaliana genome (TAIR10) using Bowtie 1 with the option allowing for all possible multiple alignments without any mismatches.
Then, the number of reads that were aligned onto the annotated loci of 1) coding and non-coding genes excluding miRNA genes in TAIR10 and 2) mature miRNAs in miRBase 21 were calculated by using coverageBed of BEDTools.
For each sample, reads per million mapped reads (RPM) values of the individual loci were computed.
Genome_build: Arabidopsis_thaliana.TAIR10.31
Supplementary_files_format_and_content: Tab-delimited text file including RPM values for the individual samples

organism: Arabidopsis thaliana

source_name: Stomatal lineage cells

growth_protocol: Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used as WT in this study.  Plants were grown on 1/2 Murashige and Skoog (MS) agar media without sucrose or in soil at 22℃ under long-day (16h-light/8h-dark) conditions.

extract_protocol:
RNA immunoprecipitation experiments were conducted as previously described (Carbonell, 2012) with a minor modification.  In brief, four grams of Arabidopsis leaves from 6 day-old seedlings were ground in liquid nitrogen and dissolved in 12-ml protein lysis buffer (50mM Tris-HCl, PH 7.4, 2.5mM MgCl2, 100mM KCl, 0.1% Nonident P-40, 0.5mM PMSF, proteinase inhibitor cocktails (EDTA-free protease inhibitor cocktail Tablet/50ml lysis buffer, Roche), 50 units/ml RNase inhibitor (Thermo Scientific)). Cell extracts were centrifuged two times at 12,000 rcf at 4℃ for 15 min to remove cell debris. The supernatant was incubated with binding control agarose beads (Chromotek, Cat. No. bab-20) without antibody for 30min at 4℃ to remove the non-specific binding, then incubated with anti-GFP agarose beads (Chromotek, Cat. No. gta-20) for 2hr at 4℃.  The beads were washed 3 times by protein lysis buffer at 4℃.  RNA was recovered by incubating the beads in same volume of proteinase K buffer (50mM Tris-HCl, PH 7.4, 25mM EDTA, 300 mM NaCl, 1.25% SDS, 1μg/ul proteinase K (Thermo Scientific, Cat. No. AM2546)) for 15min at 65℃ and purified using the Zymo micro RNA kit (Zymo Research, Cat. No. R1061). Small RNAs were quantified using the Agilent 2100 Bioanalyzer system (Aglient small RNA Kit, Cat. No. 5067-1548).
Small RNAs were converted to cDNA libraries using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs, MA, USA).  5’- and 3’-adapters were ligated to the purified small RNA, followed by reverse transcription using Protoscript II reverse transcriptase and incorporation of index tags by PCR. The resulted libraries were subjected to size selection (145–160 bp) using Sage Pippin Prep (Sage science, MA, USA) on a 3% agarose gel.  The cDNA libraries were the sequenced using the Illumina Hiseq 2500 system, generating 50 bp single-end reads.

tag: ecotype

value: stomatral entry stage cells

tag: age

value: 6 days after germination

tag: ip antibody

value: GFP


title: Sample 22 ML1p::AGO1DAH

description:
Transgenic plant harboring ML1 promoter, GFP and AGO1DAH (slicer-defective AGO1) construct
Sample 22

tag: cell type

value: epidermal cells


title: Sample 19 FAMAp::AGO1DAH

description:
Transgenic plant harboring FAMA promoter, GFP and AGO1DAH (slicer-defective AGO1) construct
Sample 19

value: stomatal differentration stage cells


title: Sample 20 EPF1p::AGO1DAH

description:
Transgenic plant harboring EPF1 promoter, GFP and AGO1DAH (slicer-defective AGO1) construct
Sample 20


title: Treat_082_RBG19177

description: Treated_ICGScluster6_006_HSCs (CD31+kit+CD45-)

tag: treatment


title: Treat_083_RBG19189

description: Treated_ICGScluster3_010_HSCs (CD31+kit+CD45-)

tag: icgs cluster


title: Treat_081_RBG19165

description: Treated_ICGScluster2_028_HSCs (CD31+kit+CD45-)


title: Treat_080_RBG19153

description: Treated_ICGScluster3_009_HSCs (CD31+kit+CD45-)


title: Sample 18 MUTEp::AGO1DAH

description:
Transgenic plant harboring MUTE promoter, GFP and AGO1DAH (slicer-defective AGO1) construct
Sample 18

value: stomatal commitment stage cells


title: Sample 13 EPF2p::AGO1DAH

description:
Transgenic plant harboring EPF2 promoter, GFP and AGO1DAH (slicer-defective AGO1) construct
Sample 13


title: Treat_078_RBG19009

description: Treated_ICGScluster1_028_HSCs (CD31+kit+CD45-)


title: Treat_079_RBG19021

description: Treated_ICGScluster1_029_HSCs (CD31+kit+CD45-)


title: Sample 10 MUTEp::AGO1DAH

description:
Transgenic plant harboring MUTE promoter, GFP and AGO1DAH (slicer-defective AGO1) construct
Sample 10


title: Sample 14 ML1p::AGO1DAH

description:
Transgenic plant harboring ML1 promoter, GFP and AGO1DAH (slicer-defective AGO1) construct
Sample 14


title: Treat_086_RBG19022

description: Treated_ICGScluster3_012_HSCs (CD31+kit+CD45-)


title: Treat_077_RBG18997

description: Treated_ICGScluster7_005_HSCs (CD31+kit+CD45-)


title: Treat_073_RBG18937

description: Treated_ICGScluster2_027_HSCs (CD31+kit+CD45-)


title: Treat_074_RBG18949

description: Treated_ICGScluster3_008_HSCs (CD31+kit+CD45-)


title: Treat_075_RBG18961

description: Treated_ICGScluster1_026_HSCs (CD31+kit+CD45-)


title: Sample 9 SPCHp::AGO1DAH

description:
Transgenic plant harboring SPCH promoter, GFP and AGO1DAH (slicer-defective AGO1) construct
Sample 9


title: Sample 5 EPF2p::AGO1DAH

description:
Transgenic plant harboring EPF2 promoter, GFP and AGO1DAH (slicer-defective AGO1) construct
Sample 5


title: Sample 17 SPCHp::AGO1DAH

description:
Transgenic plant harboring SPCH promoter, GFP and AGO1DAH (slicer-defective AGO1) construct
Sample 17


title: Sample 4 EPF1p::AGO1DAH

description:
Transgenic plant harboring EPF1 promoter, GFP and AGO1DAH (slicer-defective AGO1) construct
Sample 4


title: Treat_072_RBG19200

description: Treated_ICGScluster2_026_HSCs (CD31+kit+CD45-)


title: Treat_070_RBG19176

description: Treated_ICGScluster1_025_HSCs (CD31+kit+CD45-)


title: Treat_071_RBG19188

description: Treated_ICGScluster2_025_HSCs (CD31+kit+CD45-)


title: Sample 2 MUTEp::AGO1DAH

description:
Transgenic plant harboring MUTE promoter, GFP and AGO1DAH (slicer-defective AGO1) construct
Sample 2


title: Sample 3 FAMAp::AGO1DAH

description:
Transgenic plant harboring FAMA promoter, GFP and AGO1DAH (slicer-defective AGO1) construct
Sample 3


title: Sample 11 FAMAp::AGO1DAH

description:
Transgenic plant harboring FAMA promoter, GFP and AGO1DAH (slicer-defective AGO1) construct
Sample 11


title: Sample 12 EPF1p::AGO1DAH

description:
Transgenic plant harboring EPF1 promoter, GFP and AGO1DAH (slicer-defective AGO1) construct
Sample 12


title: Sample 1 SPCHp::AGO1DAH

description:
Transgenic plant harboring SPCH promoter, GFP and AGO1DAH (slicer-defective AGO1) construct
Sample 1


title: mouse_BAT_siPERK24hr

city: Miyazaki City

first: Hideki

last: Nishitoh

state: Miyazaki Prefecture

address: 5200 Kihara, Kiyotake

department: Department of Medical Sciences

country: Japan

institute: University of Miyazaki

data_processing:
Illumina Casava1.8 software used for basecalling.
Sequenced reads were mapped to hg19 whole genome using tophat2 v2.0.13 with default parameters.
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using cufflinks v2.2.1.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include RPKM values

source_name: Primary brown adipocyte

extract_protocol:
mRNAs were isolated from total RNA by NEBNEXT Poly(A) mRNA Magnetic Isolation Module (NEBE7490).
NEBNext mRNA Library Prep Master Mix Set for Illumina (E6110) was used with 250 ng of mRNA for the construction of sequencing libraries according to the manufacturer's protocol.

value: Primary brown adipocyte

value: siPERK

tag: passage


title: mouse_BAT_sicont24hr

value: sicontrol


title: Treat_069_RBG19164

description: Treated_ICGScluster3_007_HSCs (CD31+kit+CD45-)


title: Treat_065_RBG19008

description: Treated_ICGScluster1_023_HSCs (CD31+kit+CD45-)


title: Treat_067_RBG19128

description: Treated_ICGScluster2_024_HSCs (CD31+kit+CD45-)


title: Treat_076_RBG18985

description: Treated_ICGScluster1_027_HSCs (CD31+kit+CD45-)


title: Treat_066_RBG19020

description: Treated_ICGScluster6_005_HSCs (CD31+kit+CD45-)


title: Treat_064_RBG18996

description: Treated_ICGScluster1_022_HSCs (CD31+kit+CD45-)


title: Treat_068_RBG19152

description: Treated_ICGScluster1_024_HSCs (CD31+kit+CD45-)


title: Sample 6 ML1p::AGO1DAH

description:
Transgenic plant harboring ML1 promoter, GFP and AGO1DAH (slicer-defective AGO1) construct
Sample 6


title: mouse_BAT_siPERK16hr


title: mouse_BAT_sicont16hr


title: Treat_063_RBG18972

description: Treated_ICGScluster1_021_HSCs (CD31+kit+CD45-)


title: Treat_062_RBG18960

description: Treated_ICGScluster1_020_HSCs (CD31+kit+CD45-)


title: HEK293 U6 snRNA RiboMeth-Seq replicates 1-3

city: Regensburg

first: Gerhard

last: Lehmann

address: Universitaetsstrasse 31

department: Biochemistry 1

country: Germany

institute: University of Regensburg

description:
small RNA sequencing data
RMS_LARP7_ko_U6.xlsx

data_processing:
Base calling for HiSeq runs were performed using CASAVA 1.8.2 software
Base calling for MiSeq runs were performed using MiSeq Control Software 2.6.2.1
Adapter trimming was performed using cutadapt version 1.17 with parameters --overlap=8 --minimum-length=15 --discard-untrimmed
Mapping against U6 and U2 fasta sequences were performed using Bowtie 2 version 2.3.4.1
Further analysis were performed in R version 3.3.3 using the ShortRead 1.32.1 package according to Birkedal et. al. "Profiling of Ribose Methylations in RNA by High-Throughput Sequencing". Angew Chem Int Ed Engl. 2015 Jan 7;54(2):451-5
Genome_build: hg38
Supplementary_files_format_and_content: Excel files with RiboMeth-seq scores A and B

source_name: HEK293 wildtype

growth_protocol: HEK293 cells were cultivated in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. Cells were grown under standard conditions at 37°C in 5% CO2 atmosphere.

extract_protocol:
Total RNA was extracted with TRIzol reagent an the RNAs of interest were purified by two consecutive pulldowns with a mix of three biotinylated antisense oligonucleotides and dynabeads M-270 streptavidin (Invitrogen).
RNA samples purified by antisense oligonucleotide pulldowns were fragmented by alkaline hydrolysis in 50 mM Na2CO3 pH 9.0 for 50-55 minutes at 90°C. RNA fragments were resolved on polyacrylamide (acrylamid/bisacrylamid 19:1) urea gels, stained with SYBR gold and gel slices containing RNA fragments in the range between 21 nt and 35 nt were excised. Upon elution and precipitation of the fragments, the RNA was treated with 0.5 U/ml calf intestinal alkaline phosphatase (NEB) for 1 h at 37°C. Dephosphorylated RNAs were purified by phenol/chloroform/isoamyl alcohol extraction followed by precipitation. Phosphorylation of the 5’ ends occurred with the T4 polynucleotide kinase (Thermo Scientific), followed by heat-inactivation of the enzyme, removal of unincorporated ATPs via Illustra MicroSpin G-25 gel filtration columns, phenol/chloroform/isoamyl alcohol extraction of the RNA fragments and precipitation. Small RNA libraries were generated by ligating an adenylated adapter, [5’-App-TGGAATTCTCGGGTGCCAAGG-(C7-amino)-3’], to the 3’ end of the RNA fragments by a truncated T4 RNA Ligase 2. Ligated fragments were gel purified prior to ligation to the 5' RNA adapter (5'-GUUCAGAGUUCUACAGUCCGACGAUC-3') using the T4 RNA Ligase 1 (NEB). After reverse-transcription with a specific primer, the cDNA was amplified by PCR, gel-fractionated and the region above empty adapter dimers was cut out. PCR products from these gel slices were eluted in elution buffer, precipitated and dissolved in water. The quality of the libraries was assessed by measurement on a TapeStation 4200 device.

tag: origin

value: human embryonic kidney

tag: purification

value: U6 snRNA enriched by pulldown with biotinylated antisense oligonucleotides from total RNA


title: HEK293 LARP7 ko U6 snRNA RiboMeth-Seq replicates 1-4

source_name: HEK293 LARP7 knockout


title: a-LARP7 IP

description: small RNA sequencing data

data_processing:
Base calling was performed using CASAVA 1.8.2 software.
Adapter trimming was performed using cutadapt version 1.17 with parameters --overlap=5 --minimum-length=16.
Mappings were performed using Bowtie 2 version 2.3.4.1 against Homo_sapiens.GRCh38.dna_sm.primary_assembly.fa (ftp://ftp.ensembl.org/pub/release-92/fasta/homo_sapiens/dna/).
Further analysis was performed in R version 3.3.3 using the packages ShortRead 1.32.1, rtracklayer 1.34.2, Biostrings 2.42.1, GenomicRanges 1.26.4, GenomicAlignments 1.10.1, DESeq2 1.14.1 and openxlsx 4.1.0.
Annotation data were retrieved from Ensembl (ensembl.hsa.GRCh38.92.gtf), UCSC table browser (RepeatMasker, tRNA, snaR; status February 2018), snoatlas.bioinf.uni-leipzig.de (snoRNA; status July 2018).
"Pol I" rRNAs were combined from Ensembl and RepeatMasker annotations and additional rRNA annotations were retrieved from NCBI GRCh38 (status July 2018).
The category "Pol III" was created by combining the following annotations: 5S-rRNA from Ensembl and RepeatMasker, tRNA from RepeatMasker and UCSC, U6, 7SL, 7SK and scRNA from Ensembl and RepeatMasker, vaultRNA, RMRP and RNaseP_nuc (RPPH1) from Ensembl, and snaR from UCSC. Overlapping or multiple annotations were merged together.
SnoRNA annotation from snoRNA atlas were converted from h19 to hg38 coordinates and combined together with Ensembl snoRNAs. Overlapping and multiple annotations from these two datasets were merged together.
Several subcategories from Ensembl biotype annotations were grouped into the following categories: "Protein coding", "Pseudogene", "Long noncoding" and "RepeatMasker".
To reduce multiple counting the annotations for small RNAs (i.e., miRNAs, Pol III transcripts, snoRNAs and Pol I rRNAs) were cut out from the remaining annotations with an additional spacer of ten bases.
Overlaps between annotations and sequenced reads were counted on exon level with at least 1 base overlap.
Genome_build: hg38
Supplementary_files_format_and_content: Count table in excel format.

extract_protocol:
Polyclonal anti-LARP7 antibodies from rabbit serum SY7862 were coupled overnight at 4°C to Protein A sepharose beads. Cells were washed with cold PBS and lysed in 1.5 ml of lysis buffer [25mM Tris (pH 7.5),150mM KCl, 0.5% (v/v) NP-40, 2mM EDTA, 1mM NaF, 1mM DTT and 0.5mM AEBSF]. The lysates were cleared by centrifugation at 17,000xg for 30 min, before addition to the beads. After 3 h of rotation at 4°C, the beads were washed four times with wash buffer [50mM Tris–HCl (pH 7.5), 300mM KCl, 1mM MgCl2 and 0.5% (v/v) NP-40] and once with PBS. For RNA extraction 200 µl Proteinase K buffer [200mM Tris (pH 7.5), 300mM NaCl, 25mM EDTA, 2% SDS] containing 0.2 mg/ml Proteinase K were added. The samples were incubated 30 min at 50°C, the RNA was then isolated with phenol/chloroform/isoamyl alcohol. Input samples were prepared by extracting total RNA directly from an aliquot of the lysates using TRIzol according to the manufacturer’s instructions.
In short, an adenylated adapter [5’-App-TGGAATTCTCGGGTGCCAAGG-(C7-amino)-3’] was ligated to the 3’ end of the total RNA and RNA co-precipitated in anti-LARP7 immunoprecipitations by a truncated T4 RNA Ligase 2. Subsequently, the 5’ RNA-adapter (5’-GUUCAGAGUUCUACAGUCCGACGAUC-3’) was ligated using the T4 RNA Ligase 1 (NEB). After reverse-transcription using a specific primer, the cDNA was amplified by PCR, gel-fractionated and bands or regions above empty adapter dimers were cut out. PCR products from these gel slices were eluted in elution buffer, precipitated and dissolved in water. The quality of the libraries was assessed by Bioanalyzer measurements.

tag: affinity purification

value: by anti-LARP7 immunoprecipitation


title: Treat_061_RBG18948

description: Treated_ICGScluster1_019_HSCs (CD31+kit+CD45-)

value: Hematopoietic stem cells (CD31+kit+CD45-)


title: Treat_060_RBG18936

description: Treated_ICGScluster2_023_HSCs (CD31+kit+CD45-)


title: Treat_059_RBG19199

description: Treated_ICGScluster1_018_HSCs (CD31+kit+CD45-)


title: [ChIP-seq, FLAG] MEF flagAscl1 IP (rep3)

description: TGM_flagAscl1_A1allinput.idrpeaks.0.1.merged.bed

value: Mouse embryonic fibroblasts

value: flagAscl1 + rtTA

value: 2 days

tag: chip antibody


title: HEK293 Input

description: small RNA sequencing data


title: RM Input DNA

city: Grand Rapids

first: Russell

last: Jones

state: Michigan

address: 333 Bostwick Ave. NE

country: USA

institute: Van Andel Institute

description: RM Input DNA

data_processing:
Read were mapped using STAR
Peaks were called using MACS2.0
Log2ChIP/Input was computed using DeepTools2.0
Genome_build: mm10
Supplementary_files_format_and_content: Log2 Ratio (ChIP/ Input)

source_name: CD4+ Th17 cells

growth_protocol: CD4+ T cells were activated using plate-bound anti-CD3 (5µg/ml) and anti-CD28 (2µg/ml). Cells were cultured in Th17-polarizing medium containing IL-6 (20ng/ml), TGF-β (2ng/ml), anti-IL-4 (2.5µg/ml), anti-IFN-γ (2.5µg/ml), and anti-IL-2 (2.5µg/ml) for 5 days. Control medium contained 200 uM methionine and methionine restriction (MR) conditions contained (3 uM).

extract_protocol:
Aliquots of 100,000 Th17 cells were thawed on ice for 5 min and immediately resuspended in 20uL of EZ Nuclei Isolation Buffer (Sigma-Aldrich), and incubated for 5 min on ice. Samples were digested using 2 Units/uL MNase in 20uL MNase Digestion Buffer (NEB) for 5 minutes at 37oC. Reactions were stopped by the addition of 10mM EDTA and 0.1% Trition/0.1% Deoxycholate (final concentration). Isolated chromatin was incubated on ice for 15 min, followed by vortexing on medium setting for 30 sec. The volume was adjusted to 200uL with Complete IP Buffer (20mM Tris-HCL pH 8.0, 2mM EDTA, 150mM NaCl, 0.1% Triton X- 100, 10mM Sodium Butyrate, 1x Protease Inhibitor Cocktail, 1mM PMSF), and incubated for 1 h at 4oC on a gentle rocking platform. 10% of the total chromatin was removed to assess digestion efficiency and to use as an input control. Chromatin for immunoprecipitation was pre-cleared using Protein A Dynabeads (Invitrogen) for 1–4 hours at 4oC and subjected to IP overnight at 4oC (0.03μg H3K4me3 Abcam ab8580; 0.2μg H3K4me1 Abcam ab8895). Bead-chromatin complexes were washed using low-salt wash buffer (0.1% SDS, 1% Trition X-100, 2mM EDTA, 20mM Tris pH 8.0, 150mM NaCl, 1x Protease Inhibitor Cocktail, 10mM Sodium Butyrate), high-salt wash buffer (0.1% SDS, 1% Trition X-100, 2mM EDTA, 20mM Tris pH 8.0, 500mM NaCl, 1x Protease Inhibitor Cocktail, 10mM Sodium Butyrate). Chromatin was eluted from the beads using elution buffer (1% SDS, 100mM NaHCO3) by shaking for 1 h at 65oC. DNA was then purified by phenol-chloroform extraction using Maxtract tubes (Qiagen) and then ethanol precipitated overnight.
Immunoprecipitated DNA was prepared for sequencing on the Illumina platform using the NEBNext ChIP-Seq Library Prep Master Mix Set using modified Illumina TruSeq adapters.

tag: cell subtype

value: CD4+ T-cells

tag: strain

value: Cultured T-cells

tag: antibody


title: Treat_058_RBG19187

description: Treated_ICGScluster2_022_HSCs (CD31+kit+CD45-)


title: Ctrl Input DNA

description: Ctrl Input DNA


title: Treat_057_RBG19175

description: Treated_ICGScluster1_017_HSCs (CD31+kit+CD45-)


title: RM H3K3me1

description: RM H3K3me1

value: 0.2microg H3K4me1 Abcam ab8895


title: Treat_055_RBG19151

description: Treated_ICGScluster6_004_HSCs (CD31+kit+CD45-)


title: Treat_056_RBG19163

description: Treated_ICGScluster1_016_HSCs (CD31+kit+CD45-)


title: MR  RNA-seq rep3

description:
CD4+ T-cells differentiated with TGF-beta and IL-6 for 5 days in medium with 3 uM methionine
Th17_3uM_rep_3

data_processing:
Data were sequenced on HiSeq 2500 instrument (Illumina, San Diego, CA) using 41bpX18bp pair-end sequencing. Second mate was used for sample demultiplexing, at which point individual single-end fastqs were aligned to mm10 genome using STAR with following options --outFilterMultimapNmax 15 --outFilterMismatchNmax 6 --outReadsUnmapped Fastx --outSAMstrandField All. Gene expression was obtained using in-house scripts based on MACS and HTSeq (https://github.com/ctlab/quant3p).
Genome_build: mm10
Supplementary_files_format_and_content: Table containing CPM counts for all samples and gene fold change expression, P-value , FDR

extract_protocol:
RNA was extacted using Trizol following manufacturer’s protocol.
For cDNA synthesis, custom oligo-dT primers were used with a barcode and adapter-linker sequence (CCTACACGACGCTCTTCCGATCT—XXXXXXXX-T15). After first strand synthesis, samples were pooled together based on Actb qPCR values, and RNA-DNA hybrids degraded using consecutive acid-alkali treatment. A second sequencing linker (AGATCGGAAGAGCACACGTCTG) was ligated using T4 ligase (NEB) followed by SPRI clean-up. The mixture was then PCR enriched for 12 cycles and SPRI purified to yield final strand specific RNA-seq libraries as previously described (Jha et al., 2015)
Library strategy: Libraries were sequenced using a HiSeq 2500 (Illumina) using 40 bp by 10 bp single-end sequencing.

tag: agent

value: 3 uM methionine


title: Treat_052_RBG19007

description: Treated_ICGScluster2_021_HSCs (CD31+kit+CD45-)


title: Ctrl  RNA-seq rep3

description:
CD4+ T-cells differentiated with TGF-beta and IL-6 for 5 days in medium with 200 uM methionine
Th17_Full_rep_3

value: 200 uM methionine


title: Treat_054_RBG19139

description: Treated_ICGScluster3_006_HSCs (CD31+kit+CD45-)


title: Treat_053_RBG19019

description: Treated_ICGScluster1_015_HSCs (CD31+kit+CD45-)


title: MR  RNA-seq rep2

description:
CD4+ T-cells differentiated with TGF-beta and IL-6 for 5 days in medium with 3 uM methionine
Th17_3uM_rep_2


title: Ctrl H3K3me1

description: Ctrl H3K3me1


title: Ctrl H3K3me3

description: Ctrl H3K3me3

value: 0.03microg H3K4me3 Abcam ab8580


title: Treat_050_RBG18983

description: Treated_ICGScluster1_014_HSCs (CD31+kit+CD45-)


title: RM H3K3me3

description: RM H3K3me3


title: MR  RNA-seq rep1

description:
CD4+ T-cells differentiated with TGF-beta and IL-6 for 5 days in medium with 3 uM methionine
Th17_3uM_rep_1


title: Treat_051_RBG18995

description: Treated_ICGScluster6_003_HSCs (CD31+kit+CD45-)


title: Ctrl  RNA-seq rep1

description:
CD4+ T-cells differentiated with TGF-beta and IL-6 for 5 days in medium with 200 uM methionine
Th17_Full_rep_1


title: Ctrl  RNA-seq rep2

description:
CD4+ T-cells differentiated with TGF-beta and IL-6 for 5 days in medium with 200 uM methionine
Th17_Full_rep_2


title: Treat_048_RBG18959

description: Treated_ICGScluster7_004_HSCs (CD31+kit+CD45-)


title: Treat_049_RBG18971

description: Treated_ICGScluster2_020_HSCs (CD31+kit+CD45-)


title: Tumor cells, T/P 4

city: New York

first: Yu-Jui

last: Ho

state: New York

address: 417 E 68th St

department: Cancer Biology & Genetics Program

institute: Memorial Sloan Kettering Cancer Center

data_processing:
Illumina read quality was checked for each sample using FastQC v0.11.5.
Raw fastq files were analyzed by removing adaptor sequences using Trimmomatic (0.36), aligned to the mouse genome using STAR (2.5.3a), and quantified transcript counts using featureCounts (1.6.3).
Genome_build: Reads were aligned to the mouse transcriptome (mm10, GRCm38)
Supplementary_files_format_and_content: Tab-delimited text file includes raw count table for each sample

source_name: tumor cells from KPCmut transplant PDAC tumors

growth_protocol: To generate orthotopic PDAC tumors, 5x10e5 KrasG12D/+;Trp53R172H/wt (KPCmut) PDAC cells expressing luciferase and GFP (LUC-GFP) were orthotopically transplanted into the pancreas of 8-10 week old C57BL/6 female mice as described above. Mice were monitored for tumor development (5-8 days post-transplantation) by bioluminescence imaging (BLI) on a Xenogen IVIS Spectrum imager (PerkinElmer) before being randomized into various study cohorts.

extract_protocol:
Total RNA was extracted using the RNeasy Mini Kit (Qiagen)
PolyA mRNA was selected using beads coated with polyT oligonucleotides. Purified polyA mRNA was subsequently fragmented, and first and second strand cDNA synthesis performed using standard Illumina mRNA TruSeq library preparation protocols. Double stranded cDNA was subsequently processed for TruSeq dual-index Illumina library generation.
For sequencing, pooled multiplexed libraries were run on a HiSeq 2500 machine on RAPID mode. Approximately 10 million 76bp single-end reads were retrieved per replicate  condition. Resulting RNA-Seq data was analyzed by removing adaptor sequences using Trimmomatic (29), aligning sequencing data to GRCh37.75(hg19) with STAR (30), and genome wide transcript counting using HTSeq (31) to generate a RPKM matrix of transcript counts.

treatment_protocol: PDAC-bearing mice were treated with vehicle or combined trametinib (1 mg/kg body weight), and palbociclib (100 mg/kg body weight) per os for 4 consecutive days followed by 3 days off treatment for 10 days before sacrifice.

value: tumor cells

value: T/P


title: Tumor cells, T/P 3


title: Treat_046_RBG18935

description: Treated_ICGScluster2_019_HSCs (CD31+kit+CD45-)


title: Tumor cells, T/P 2


title: Tumor cells, T/P 1


title: Ctrl_089_RBG19198

description: Ctrl_ICGScluster4_002_HSCs (CD31+kit+CD45-)

value: Ctrl


title: Ctrl_090_RBG19210

description: Ctrl_ICGScluster5_004_HSCs (CD31+kit+CD45-)


title: Tumor cells, Vehicle 4

value: Vehicle


title: Ctrl_087_RBG19174

description: Ctrl_ICGScluster6_003_HSCs (CD31+kit+CD45-)


title: Tumor cells, Vehicle 3


title: Tumor cells, Vehicle 2


title: Ctrl_088_RBG19186

description: Ctrl_ICGScluster1_042_HSCs (CD31+kit+CD45-)


title: Tumor cells, Vehicle 1


title: Ctrl_084_RBG19138

description: Ctrl_ICGScluster2_027_HSCs (CD31+kit+CD45-)


title: Ctrl_085_RBG19150

description: Ctrl_ICGScluster1_041_HSCs (CD31+kit+CD45-)


title: Treat_045_RBG19018

description: Treated_ICGScluster5_002_HSCs (CD31+kit+CD45-)


title: Treat_044_RBG19006

description: Treated_ICGScluster6_002_HSCs (CD31+kit+CD45-)


title: Ctrl_086_RBG19162

description: Ctrl_ICGScluster2_028_HSCs (CD31+kit+CD45-)


title: Ctrl_083_RBG19126

description: Ctrl_ICGScluster7_004_HSCs (CD31+kit+CD45-)


title: Treat_043_RBG18994

description: Treated_ICGScluster1_013_HSCs (CD31+kit+CD45-)


title: Treat_047_RBG18947

description: Treated_ICGScluster4_003_HSCs (CD31+kit+CD45-)


title: Stimulated_6h_2

city: Paris

first: Philippe

last: Bousso

address: 25 Rue du Docteur Roux

department: Immunology department

country: France

institute: Institut Pasteur

description: Eµ-myc B cell lymphoma

data_processing:
Quality control was performed using Picard and RSeQC.
The reads were aligned using STAR (version 2.5.2a) with default parameters.
Genes were annotated in GenCode vM16 and were quantified with featureCounts (version 1.4.3-p1).
Genome_build: mm10
Supplementary_files_format_and_content: Tab-delimited text files include count values for each sample.

molecule: polyA RNA

source_name: Eµ-myc cell

growth_protocol: Cells were cultured in RPMI 1640 (Gibco) supplemented with 10% FCS.

extract_protocol:
Cells were harvested and RNA extracted using Rneasy mini kit from Qiagen according to manufacturer’s instructions.
RNA sequencing libraries were prepared using the TruSeq stranded mRNA library preparation kit in accordance to the manufacturer instructions. Briefly 500ng of total RNA was used for purification and fragmentation of mRNA. Purified mRNA underwent first and second strand cDNA synthesis. cDNA was then adenylated, ligated to Illumina sequencing adapters and amplified by PCR using 10 cycles. Final libraries were evaluated using fluorescent based assays including PicoGreen (Life Technologies) or Qubit Fluorometer (Invitrogen) and Fragment Analyzer (Advanced Analytics) or BioAnalyzer (Agilent 2100) and were sequenced on an Illumina HiSeq2500 sequencer (v4 chemistry) using 2*125bp cycles.

treatment_protocol: Cells were either left untreated or stimulated with 5ng/mL of recombinant mouse IFNg. Stimulation was blocked at different time points through addition of 50µg/mL anti mouse-IFNg neutralising antibody (clone XMG1.2).

value: E<mu>-myc B cell lymphoma

tag: stimulation

value: IFNg


title: Stimulated_24h_3

description: Eµ-myc B cell lymphoma


title: Stimulated_1h_3

description: Eµ-myc B cell lymphoma


title: Stimulated_6h_1

description: Eµ-myc B cell lymphoma


title: Stimulated_24h_1

description: Eµ-myc B cell lymphoma


title: Stimulated_6h_3

description: Eµ-myc B cell lymphoma


title: Stimulated_24h_2

description: Eµ-myc B cell lymphoma


title: Treat_042_RBG18982

description: Treated_ICGScluster1_012_HSCs (CD31+kit+CD45-)


title: Stimulated_1h_1

description: Eµ-myc B cell lymphoma


title: Stimulated_1h_2

description: Eµ-myc B cell lymphoma


title: Treat_040_RBG18958

description: Treated_ICGScluster1_011_HSCs (CD31+kit+CD45-)


title: Unstimulated_3

description: Eµ-myc B cell lymphoma


title: Treat_041_RBG18970

description: Treated_ICGScluster2_018_HSCs (CD31+kit+CD45-)


title: Treat_038_RBG18934

description: Treated_ICGScluster1_010_HSCs (CD31+kit+CD45-)


title: R5 mac

type: RNA

city: Heidelberg

first: Katrin

last: Deumelandt

address: INF 280

department: DKTK Clinical Cooperation Unit Neuroimmunology and Brain Tumor Immunology

institute: German Cancer Research Center (DKFZ)

hyb_protocol: Followed NanoString nCounter XT gene expression assay protocol. Briefly, hybridization buffer was mixed with the nCounter Mouse Immunology Panel reporter codeset to prepare a master mix. Master mix, sample and Rnase-fee water was combined with nCounter Mouse Immunology Panel Capture codeset. Samples with 25ng total RNA were then incubated for 20 h at 65 deg C.

scan_protocol: Raw counts of mRNA hybridized to oligos were counted using the NanoString nCounter SPRINT instrument.

data_processing: Controls and low count genes were removed from the nanostring count matrix, followed by a scalar normalization and variance modeling as previously described (Robinson MD et al., Genome Biol. 2010;  Law CW et al., Genome Biol. 2014). Differential gene expression was performed by an eBayes adjusted moderted t-statistic linear regression model (Ritchie ME et al., Nucleic Acids Res. 2015).

label: Capture probe-3'biotin, Reporter probe-5' barcode signal

extract_protocol: Cells were isolated from Gl261 tumors by enzymatic digestion and myelin removal by density gradient centrifugation. CD45highCD11b+ cells were purified by fluorescence activated cell sorting. RNA was extracted using the Arcturus PicoPure RNA Isolation Kit (Thermo Fisher)

treatment_protocol: For immune checkpoint therapy, 100 µg per mouse anti-CTLA-4 (9D9, BioXcell) and 250 µg per mouse anti-PD-1 (RMP1-14, BioXcell) were administered by intraperitoneal injection in 200 µl PBS on day 13, 16 and 19 after Gl261 tumor inoculation.

tag: gender

value: female

tag: therapy

value: Gl261-infiltrating CD11b+ cells (CD45highCD11b+)

tag: phenotype

value: PD-1 and CTLA-4 responder


title: R4 mac

label_protocol: Followed NanoString nCounter XT gene expression assay protocol.


title: R3 mac


title: Unstimulated_2

description: Eµ-myc B cell lymphoma


title: Ctrl_082_RBG19209

description: Ctrl_ICGScluster1_040_HSCs (CD31+kit+CD45-)

data_processing:
RNA-seq reads were aligned to mm10 using GSNAP (version 2015-09-29) with parameters (-B 5 -t 24 -n 1 -Q -N 1).
Reads in features were counted with htseq-count (HTSeq version 0.6.0) with the parameter (-s no).
Genome_build: mm10
Supplementary_files_format_and_content: HT-Seq counts / gene counts (.txt)

extract_protocol:
CD31+kit+CD45- cells were FACS-purified.
Cells were sorted in 2.3 µl of lysis buffer containing 0.2% Triton X-100 (Sigma-Aldrich) and 1U of Superase-In RNase Inhibitor (Ambion) and processed following the Smart-Seq2 protocol.
Following preamplification, all samples were purified using Ampure XP beads (Beckman Coulter) at a ratio of 1:0.6. The cDNA was then quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher) and size distributions were checked on high-sensitivity DNA chips (Agilent Bioanalyzer).
Libraries were prepared using the Illumina Nextera XT DNA preparation kit.
Single-cell RNA-seq analysis was performed as described previously (Picelli et al., 2014).
Pooled libraries were run on the Illumina Hi-Seq 4000.
Samples were used to construct Nextera XT libraries (Illumina) from 125 pg of preamplified cDNA. Libraries were purified and size selected (0.5X-0.7X) using Ampure XP beads. Then, libraries were quantified using KAPA qPCR quantification kit (KAPA Biosystems).

value: Hematopoietic stem cells (CD31+kit+CD45-)


title: R1 mac


title: [RNA-seq] MEF flagMyod1 (rep2)

data_processing:
Reads were trimmed using Prinseq 0.20.4 using the following parameters: min_len=30, trim_qual_right=25, trim_left=15, lc_method=entropy, lc_threshold=65.
Trimmed reads are aligned to the mm10 reference sequence using TopHat 2.1.1 with these parameters: r=100, mate-std-dev=50, no-coverage-search.
Expression levels of RefSeq annotated genes were calculated using Cufflinks 2.2.1 with this settings: max-bundle-frags=10000000, library-type=fr-firststrand.
Genome_build: mm10
Supplementary_files_format_and_content: Files are genes.fpkm_tracking output from Cufflinks

extract_protocol:
Total RNA was extracted at 48h post dox-induction using Trizol (Invitrogen; 15596-018), following the supplier’s protocols.
Stranded mRNA libraries were prepared using the TruSeq Stranded mRNA Library Prep Kit (Illumina RS-122). Paired-end sequencing reads (2x100bp) were generated on HiSeq 2500 Illumina platforms.

value: Mouse embryonic fibroblasts

tag: transgenes

value: flagMyod1 + rtTA

tag: days post transgene induction

value: 2 days


title: Unstimulated_1

description: Eµ-myc B cell lymphoma

tag: cell type


title: NR5 mac

value: PD-1 and CTLA-4 non-responder


title: Ctrl_079_RBG19173

description: Ctrl_ICGScluster1_038_HSCs (CD31+kit+CD45-)

tag: treatment

tag: icgs cluster


title: [ChIP-seq, FLAG] MEF flagMyod1 input (rep2)

description: TGM_flagMyoD_MDallinput.idrpeaks.0.1.merged.bed

value: input


title: NR4 mac


title: Ctrl_080_RBG19185

description: Ctrl_ICGScluster2_026_HSCs (CD31+kit+CD45-)


title: [RNA-seq] MEF flagMyod1 (rep1)


title: Treat_039_RBG18946

description: Treated_ICGScluster2_017_HSCs (CD31+kit+CD45-)


title: NR3 mac


title: Ctrl_078_RBG19161

description: Ctrl_ICGScluster1_037_HSCs (CD31+kit+CD45-)


title: Ctrl_077_RBG19149

description: Ctrl_ICGScluster2_025_HSCs (CD31+kit+CD45-)


title: [ChIP-seq, FLAG] MEF flagMyod1 input (rep1)


title: [ChIP-seq, FLAG] MEF flagMyod1 IP (rep2)

value: FLAG-conjugated beads (Sigma F2426)


title: Ctrl_076_RBG19137

description: Ctrl_ICGScluster3_007_HSCs (CD31+kit+CD45-)


title: [ChIP-seq, FLAG] MEF flagMyod1 IP (rep1)


title: Ctrl_081_RBG19197

description: Ctrl_ICGScluster1_039_HSCs (CD31+kit+CD45-)


title: [ChIP-seq, FLAG] MEF flagAscl1 input (rep2)

description: TGM_flagAscl1_A1allinput.idrpeaks.0.1.merged.bed

value: flagAscl1 + rtTA


title: Treat_037_RBG19017

description: Treated_ICGScluster1_009_HSCs (CD31+kit+CD45-)


title: Treat_036_RBG19005

description: Treated_ICGScluster7_003_HSCs (CD31+kit+CD45-)


title: [ChIP-seq, FLAG] MEF flagAscl1 input (rep1)


title: AdjacentNormalStroma_patient2 [Stroma_2N]

city: Beijing

first: Rongfang

last: Shen

address: No. 17 South Panjiayuan, Chaoyang District

department: National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital

institute: Chinese Academy of Medical Sciences and Peking Union Medical College

hyb_protocol: 0.72 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 30ul containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 30ul of 2x Agilent GE hybridization buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Hmn GE 8×60K V2 Microarrays (G4851B) for 17 hours at 65 °C in a rotating Agilent hybridization oven.  After hybridization, microarrays were washed 1 minute twice at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37 °C GE Wash buffer 2 (Agilent), then dried immediately.

scan_protocol: Slides were scanned immediately after washing on the Agilent Microarray Scanner System  (G2600D) using one color scan setting for 8x60k array slides (Scan Area full agilent slide, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).

data_processing: Background correction with normexp method and normalization with quantile method in  R package limma were applied to process raw data.Probes indicated by the ControlType column and expression less than one arrays above background were filtered.

source_name: Adjacent normal tissue, more than 5 cm away from tumor

label_protocol: Cyanine-3 (Cy3) labeled cRNA was prepared from 0.1 ug RNA using the LowInput QuickAmp Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA).  Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.

growth_protocol: Fresh tissues were cleaned with normal saline solution and frozen at 80°C within 30 min after  surgical resection before RNA preparation.

extract_protocol: Total RNA was extracted using the RNeasy Mini kit (Qiagen, MD, USA). The RNA was quantified using an ND-1000 UV-VIS Spectrophotometer (NanoDrop Technologies, DE, USA), and its integrity was assessed using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). An RNA with integrity number (RIN) greater than 7.0 was used in this study.

treatment_protocol: Stroma compartment of cancer and adjacent normal samples were obtained by microdissection. All colorectal tissue tissues were embedded in OCT (Thermo Fisher) and cut into slices in freezing microtome, each slice with a diameter of 10 μm, then laying freezing tissue slices in hematoxylin for 25 seconds. Next, stroma tissues were isolated with a needle under microscope and collected with Eppendorf tubes.

tag: subject status

value: rectal cancer

value: male

tag: age

tag: tissue type

value: adjacent normal tissue

tag: family history

value: no

tag: blood vessel invasion

tag: nerve invasion

tag: adjuvant chemotherapy

value: yes

tag: chemotherapy regimens

value: XELOX

tag: adjuvant radiotherapy


title: U6 snRNA RiboMeth-Seq from human blood of a sibling affected by Alazami syndrome 1

description:
Blood offsprings Alazami syndrome 1
small RNA sequencing data
processed data file: RMS_Alazami_U6_blood.xlsx
Offspring_Alazami1_blood

source_name: Blood

extract_protocol:
Human blood was collected in PAXgene Blood RNA Tubes and total RNA was extracted from pelleted material with TRIzol reagent.
Total RNA was extracted with TRIzol reagent an the RNAs of interest were purified by two consecutive pulldowns with a mix of three biotinylated antisense oligonucleotides and dynabeads M-270 streptavidin (Invitrogen).
RNA samples purified by antisense oligonucleotide pulldowns were fragmented by alkaline hydrolysis in 50 mM Na2CO3 pH 9.0 for 50-55 minutes at 90°C. RNA fragments were resolved on polyacrylamide (acrylamid/bisacrylamid 19:1) urea gels, stained with SYBR gold and gel slices containing RNA fragments in the range between 21 nt and 35 nt were excised. Upon elution and precipitation of the fragments, the RNA was treated with 0.5 U/ml calf intestinal alkaline phosphatase (NEB) for 1 h at 37°C. Dephosphorylated RNAs were purified by phenol/chloroform/isoamyl alcohol extraction followed by precipitation. Phosphorylation of the 5’ ends occurred with the T4 polynucleotide kinase (Thermo Scientific), followed by heat-inactivation of the enzyme, removal of unincorporated ATPs via Illustra MicroSpin G-25 gel filtration columns, phenol/chloroform/isoamyl alcohol extraction of the RNA fragments and precipitation. Small RNA libraries were generated by ligating an adenylated adapter, [5’-App-TGGAATTCTCGGGTGCCAAGG-(C7-amino)-3’], to the 3’ end of the RNA fragments by a truncated T4 RNA Ligase 2. Ligated fragments were gel purified prior to ligation to the 5’ RNA adapter (5’-GUUCAGAGUUCUACAGUCCGACGAUC-3’) using the T4 RNA Ligase 1 (NEB). After reverse-transcription with a specific primer, the cDNA was amplified by PCR, gel-fractionated and the region above empty adapter dimers was cut out. PCR products from these gel slices were eluted in elution buffer, precipitated and dissolved in water. The quality of the libraries was assessed by measurement on a TapeStation 4200 device.

tag: origin

value: human blood

tag: purification

value: U6 snRNA enriched by pulldown with biotinylated antisense oligonucleotides from total RNA


title: U6 snRNA RiboMeth-Seq from B-LCL cell line originating from a healthy parent

description:
B-LCL Parental replicates 1-2
small RNA sequencing data
processed data file: RMS_Alazami_U6_B-LCL.xlsx
B-LCL_Parental_rep1

source_name: Blood lymphocytes

growth_protocol: B-LCLs cells were cultivated in RPMI-1640 with L-glutamine supplemented with 15% fetal bovine serum. Cells were grown under standard conditions at 37°C in 5% CO2 atmosphere.

extract_protocol:
Total RNA was extracted with TRIzol reagent an the RNAs of interest were purified by two consecutive pulldowns with a mix of three biotinylated antisense oligonucleotides and dynabeads M-270 streptavidin (Invitrogen).
RNA samples purified by antisense oligonucleotide pulldowns were fragmented by alkaline hydrolysis in 50 mM Na2CO3 pH 9.0 for 50-55 minutes at 90°C. RNA fragments were resolved on polyacrylamide (acrylamid/bisacrylamid 19:1) urea gels, stained with SYBR gold and gel slices containing RNA fragments in the range between 21 nt and 35 nt were excised. Upon elution and precipitation of the fragments, the RNA was treated with 0.5 U/ml calf intestinal alkaline phosphatase (NEB) for 1 h at 37°C. Dephosphorylated RNAs were purified by phenol/chloroform/isoamyl alcohol extraction followed by precipitation. Phosphorylation of the 5’ ends occurred with the T4 polynucleotide kinase (Thermo Scientific), followed by heat-inactivation of the enzyme, removal of unincorporated ATPs via Illustra MicroSpin G-25 gel filtration columns, phenol/chloroform/isoamyl alcohol extraction of the RNA fragments and precipitation. Small RNA libraries were generated by ligating an adenylated adapter, [5’-App-TGGAATTCTCGGGTGCCAAGG-(C7-amino)-3’], to the 3’ end of the RNA fragments by a truncated T4 RNA Ligase 2. Ligated fragments were gel purified prior to ligation to the 5’ RNA adapter (5’-GUUCAGAGUUCUACAGUCCGACGAUC-3’) using the T4 RNA Ligase 1 (NEB). After reverse-transcription with a specific primer, the cDNA was amplified by PCR, gel-fractionated and the region above empty adapter dimers was cut out. PCR products from these gel slices were eluted in elution buffer, precipitated and dissolved in water. The quality of the libraries was assessed by measurement on a TapeStation 4200 device.


title: AdjacentNormalStroma_patient1 [Stroma_1N]

value: colon cancer

tag: gender


title: U6 snRNA RiboMeth-Seq from human blood of an healthy parent 2

description:
Blood parents 2
small RNA sequencing data
processed data file: RMS_Alazami_U6_blood.xlsx
Parental2_blood


title: U6 snRNA RiboMeth-Seq from human blood of a sibling affected by Alazami syndrome 2

description:
Blood offsprings Alazami syndrome 2
small RNA sequencing data
processed data file: RMS_Alazami_U6_blood.xlsx
Offspring_Alazami2_blood


title: TumorStroma_patient4 [Stroma_4T]

source_name: rectal cancer patient

value: female


title: AdjacentNormalStroma_patient7 [Stroma_7N]


title: TumorStroma_patient6 [Stroma_6T]

source_name: colon cancer patient


title: U6 snRNA RiboMeth-Seq from human blood of an healthy parent 1

description:
Blood parents 1
small RNA sequencing data
processed data file: RMS_Alazami_U6_blood.xlsx
Parental1_blood


title: TumorStroma_patient5 [Stroma_5T]


title: TumorStroma_patient3 [Stroma_3T]


title: TumorStroma_patient2 [Stroma_2T]


title: HEK293 T-Rex LARP7 ko stable FH-LARP7 WT rescue U6 snRNA RiboMeth-Seq replicates 1-2

description:
HEK293 T-Rex LARP7 ko LARP7 WT rescue replicates 1-2
small RNA sequencing data
processed data file: RMS_LARP7_ko_rescue_U6.xlsx
rescue_WT_rep1

source_name: HEK293 T-Rex LARP7 knockout stably expressing FH-LARP7 wildtype

growth_protocol: HEK293 T-Rex LARP7 ko cells stably expressing FH-LARP7 WT were cultivated in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin, 5 μg/mL blasticidin and 100 μg/mL hygromicin B. Cells were grown under standard conditions at 37°C in 5% CO2 atmosphere.

treatment_protocol: Induction of FH-LARP7 expression by tetracycline.

value: human embryonic kidney


title: [ChIP-seq, FLAG] MEF flagAscl1 IP (rep1)

value: Mouse embryonic fibroblasts

tag: transgenes

tag: chip antibody


title: Treat_033_RBG18969

description: Treated_ICGScluster1_008_HSCs (CD31+kit+CD45-)

source_name: the Aorta surrounded by Gonads and Mesonephros tissues (AGM)

tag: cell type

value: Hematopoietic stem cells (CD31+kit+CD45-)


title: TumorStroma_patient1 [Stroma_1T]


title: Treat_034_RBG18981

description: Treated_ICGScluster3_005_HSCs (CD31+kit+CD45-)

tag: treatment


title: U6 snRNA RiboMeth-Seq from B-LCL cell line originating from a boy affected by the Alazami syndrome

description:
B-LCL Alazami replicates 1-2
small RNA sequencing data
processed data file: RMS_Alazami_U6_B-LCL.xlsx
B-LCL_Alazami_rep1


title: HEK293 LARP7 ko U2 snRNA RiboMeth-Seq replicates 1-2

description:
small RNA sequencing data
RMS_LARP7_ko_U2.xlsx

value: U2 snRNA enriched by pulldown with biotinylated antisense oligonucleotides from total RNA


title: Distinct RNAs associated with FH-LARP7

description: small RNA sequencing data

data_processing:
Base calling was performed using MiSeq Control Software 2.6.2.1.
Adapter trimming was performed using cutadapt version 1.17 with parameters --overlap=5 --minimum-length=16.
Mappings were performed using Bowtie 2 version 2.3.4.1 against Homo_sapiens.GRCh38.dna_sm.primary_assembly.fa (ftp://ftp.ensembl.org/pub/release-92/fasta/homo_sapiens/dna/).
Further analysis was performed in R version 3.3.3 using the packages ShortRead 1.32.1, rtracklayer 1.34.2, Biostrings 2.42.1, GenomicRanges 1.26.4 and GenomicAlignments 1.10.1.
Annotation data were retrieved from Ensembl (ensembl.hsa.GRCh38.92.gtf), UCSC table browser (RepeatMasker, tRNA, snaR; status February 2018), snoatlas.bioinf.uni-leipzig.de (snoRNA; status July 2018).
"Pol I" rRNAs were combined from Ensembl and RepeatMasker annotations and additional rRNA annotations were retrieved from NCBI GRCh38 (status July 2018).
The category "Pol III" was created by combining the following annotations: 5S-rRNA from Ensembl and RepeatMasker, tRNA from RepeatMasker and UCSC, U6, 7SL, 7SK and scRNA from Ensembl and RepeatMasker, vaultRNA, RMRP and RNaseP_nuc (RPPH1) from Ensembl, and snaR from UCSC. Overlapping or multiple annotations were merged together.
SnoRNA annotation from snoRNA atlas were converted from h19 to hg38 coordinates and combined together with Ensembl snoRNAs. Overlapping and multiple annotations from these two datasets were merged together.
Several subcategories from Ensembl biotype annotations were grouped into the following categories: "Protein coding", "Pseudogene", "Long noncoding" and "RepeatMasker".
To reduce multiple counting the annotations for small RNAs (i.e., miRNAs, Pol III transcripts, snoRNAs and Pol I rRNAs) were cut out from the remaining annotations with an additional spacer of ten bases.
Overlaps between annotations and sequenced reads were counted on exon level with at least one
base overlap. Therefore, all genomic ranges for a biotype were reduced to one
object, which was then compared with the mapping positions. The category Pol
III was split into subgroups and analyzed accordingly.
Genome_build: hg38
Supplementary_files_format_and_content: Excel table with raw counts for all Bands on biotype level.

source_name: HEK293 T-REx FH-LARP7

growth_protocol: HEK293 cells were cultivated in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin, 5 μg/mL blasticidin and 100 μg/mL hygromicin B. Cells were grown under standard conditions at 37°C in 5% CO2 atmosphere.

extract_protocol:
The cells were lysed on ice for 10 min in buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl, 2.5 mM MgCl2, 1% NP-40, protease inhibitors and RNase inhibitors. The lysate was passed 6 times through a 26G needle followed by water bath sonication. After sonication, the lysates were centrifuged for 20 min at 14,000 rpm and 4°C. The pre-equilibrated ANTI-FLAG M2 affinity gel (Sigma-Aldrich) was incubated with the lysates for 3 h on a head-over-tail rotor at 4°C. After incubation, beads were collected and were washed three times with washing buffer containing 50 mM HEPES pH 7.5, 300 mM NaCl and 2.5 mM MgCl2, followed by the last wash using 1 x PBS. FH-LARP7 and the interacting RNAs were eluted using 200 µg/mL 3x FLAG peptide (Sigma-Aldrich) in PBS.
RNAs co-precipitated with FH-tagged LARP7 by anti-FLAG immunoprecipitations were purified with TRIzol reagent and separated on a denaturing polyacrylamide gel. RNAs were visualized under UV-light with the SYBR gold nucleic acid gel stain (Invitrogen) and three distinct bands were excised. The RNA was eluted overnight at 4°C in 300 mM NaCl and 2 mM EDTA and was subsequently precipitated. The RNAs were treated with the tobacco acid pyrophosphatase (Epicentre) to remove RNA cap structures. An adenylated adapter [5’-App-TGGAATTCTCGGGTGCCAAGG-(C7-amino)-3’] was ligated to the 3’ end of the RNAs by a truncated T4 RNA Ligase 2. Subsequently, the 5’ RNA-adapter (5’-GUUCAGAGUUCUACAGUCCGACGAUC-3’) was ligated using the T4 RNA Ligase 1 (NEB). After reverse-transcription using a specific primer, the cDNA was amplified by PCR, gel-fractionated and bands or regions above empty adapter dimers were cut out. PCR products from these gel slices were eluted in elution buffer, precipitated and dissolved in water. The quality of the libraries was assessed by Bioanalyzer measurements.

treatment_protocol: Induction of FH-LARP7 expression was obtained by addition of 1 μg/ml tetracycline 16 h prior harvesting the cells.

tag: affinity purification

value: by anti-FLAG immunoprecipitation


title: HEK293 U2 snRNA RiboMeth-Seq


title: Treat_032_RBG18957

description: Treated_ICGScluster3_004_HSCs (CD31+kit+CD45-)

tag: icgs cluster


title: N5

first: Seungwoo

last: Hwang

address: 52 Eoeun-dong Yuseong-gu

department: Korean Bioinformation Center

institute: Korea Research Institute of Bioscience and Biotechnology

data_processing:
FastQC was used to check the sequence quality of the 100 bp paired-end sequencing reads. Trimmomatic was used to clean the reads by removing adapter sequences, bases from the ends of the reads with quality <3, sliding windows of four bases with a mean quality <15, and reads shorter than 36 bp. The cleaned reads were aligned to the hg38 human genome with Bowtie2 using default parameters. The resulting mapped data in BAM format served as an input to subsequent differential methylation analysis.
The BAM files were inputted into the MEDIPS program. The data for chromosomes 1-22 and M were selected for analysis. Sex chromosomes were excluded to avoid potential biases arising from X chromosome inactivation in female samples. The genome was binned into adjacent windows 250 nucleotides in length. The windows with zero count for all the samples were removed from all subsequent analysis. Differential methylation analysis between the CLL and control groups was performed at the window level. The parameter for data normalization and differential methylation analysis was set to edgeR and that for multiple testing correction was set according to the Benjamini-Hochberg procedure; default values were used for all other parameters. Windows with functional divergence ratios <0.01 were deemed differentially methylated.
The window-level differential methylation analysis result from MEDIPS was inputted into the ChIPseeker program. For each window, ChIPseeker assigns a corresponding Entrez Gene and one or more of the following annotations: distal intergenic, promoter (2 kb upstream to 0.5 kb downstream of transcription start site), 5’ untranslated region, exon, intron, 3’ untranslated region, and downstream. Default values were used for all other parameters.
Genome_build: hg38
Supplementary_files_format_and_content: two tab-delimited text files: (1) window-wise differential expression statistics and counts for all 6,258,571 windows with a non-zero count in at least one sample, obtained from MEDIPS, (2) annotation of 6,258,503 windows, obtained from ChIPseeker

source_name: Bone marrow

extract_protocol:
Bone marrow buffy coats were collected from the CLL patients; the median lymphoid cell percentage was 85.75% (range, 41.60–99.00%). CD19 positive B-cells were collected from five healthy donors using magnetic bead sorting (EasySep; STEMCELL Technologies, Inc., Vancouver, Canada). Purity was confirmed using flow cytometry analysis (>95.0%). Genomic DNA was isolated using the Promega Maxwell 16 MDx Instrument.
Genomic DNA (1 µg) was then sheared to 200-400 bp using a Covaris LE220 sonicator; the fragments were then subject to methyl-CpG enrichment using the Invitrogen MethylMiner Methylated DNA Enrichment Kit, which uses a recombinant form of human MBD protein-2. The enriched methylated DNA fragments were eluted as a single enriched population with a 2,000 mM NaCl elution buffer. The eluted DNA was then used to generate libraries according to the standard Illumina protocol. Briefly, the DNA fragments were subject to end repair, A-tailing of the 3’ end, Illumina adapter ligation, size selection (aiming for 300-500 bp), PCR amplification, and validation using an Agilent Bioanalyzer. The libraries were then sequenced on the Illumina HiSeq 2000 platform.

tag: disease state

value: Healthy

value: CD19+ B-cell


title: [ChIP-seq, FLAG] MEF flagAscl1 IP (rep2)

source_name: Mouse embryonic fibroblasts

extract_protocol:
FLAG-tagged TFs were immunoprecipitated using FLAG M2 antibody conjugated beads (Sigma F2426). Approximately 60-100 million cells were used per replicate. Beads (~100uL slurry per sample) were washed thrice with IP dilution buffer (1% Triton X-100, 2mM EDTA pH8.0, 20mM Tris-HCl pH8.0, 150mM NaCl, 1mM DTT, 100uM PMSF), blocked overnight in IP dilution buffer with 0.1% BSA and 0.06% sheared salmon sperm DNA (Thermo Fisher; AM9680), then washed again three times with IP dilution buffer before use. Nuclei were isolated by incubating with cell lysis buffer (5mM HEPES pH7.9, 85mM KCl, 0.5% NP40, 100uM PMSF, protease inhibitors from Roche) for 10min on ice and centrifuged at 5000rpm for 5min at 4°C, then lysed with nuclear lysis buffer (50mM Tris-HCl pH8.0, 10mM EDTA pH8.0, 1% SDS, 100uM PMSF, protease inhibitors) for 10min on ice. Chromatin was sheared using either the Bioruptor (Diagenode) or Covaris sonicator until DNA was fragmented to 200-500bp. Sheared chromatin was diluted using 3X volume of IP dilution buffer and pre-cleared for at least 4hr at 4°C using IgG beads (Sigma A0919). 1% of pre-cleared chromatin was kept as input, and the remainder was incubated with FLAG beads overnight at 4°C. Beads were washed 8 times with IP wash buffer (20mM Tris-HCl pH8.0, 2mM EDTA pH8.0, 250mM NaCl, 1% NP40, 0.05% SDS, 100uM PMSF) and once with TE buffer with 100uM PMSF. Beads and 1% input samples were reverse cross-linked overnight in IP elution buffer (50mM NaHCO3, 1% SDS) at 65°C. Isolated DNA was RNase treated for 30min at 37°C, purified using QIAquick PCR purification columns (Qiagen) and eluted in DEPC water.
ChIP-seq libraries were prepared using the NEBNext library prep kit (NEB; E6240) following the supplier’s protocols with slight modifications. Adaptor ligation was performed using regular T4 ligase and ligase buffer. Size selection (200-500bp) was performing after adaptor ligation and PCR enrichment using 2% low-melt agarose gel (Lonza; 50080), and DNA was purified using the QiaQuick gel extraction columns from Qiagen.

value: flagAscl1 + rtTA

tag: days post transgene induction

value: 2 days

value: FLAG-conjugated beads (Sigma F2426)


title: Treat_035_RBG18993

description: Treated_ICGScluster7_002_HSCs (CD31+kit+CD45-)


title: N4


title: Treat_031_RBG18945

description: Treated_ICGScluster2_016_HSCs (CD31+kit+CD45-)


title: S5D_R1

city: Bethesda

first: Pavol

last: Genzor

state: MD

address: 6720A Rockledge Drive, Suite 100

department: ACESO

institute: Henry M Jackson Foundation

description:
gel excised dsDNA
dsDNA sequencing

data_processing:
See methods section for processing steps and software versions
Genome_build: custom genome was generated using the plasmid sequence
Supplementary_files_format_and_content: fasta files; (1).unique sequences that contained casposon sequence fragment (GAGTTACCTATATCCC) were extracted and trimed to leave 3` fragemnts. Multiplicity information is retained in the seqeunce identifier after letter M. (2) Fasta file of the plasmid from which genome for alignment was generated (pUC16_TSDplus30).

organism: synthetic construct

extract_protocol:
The linear integration producs (~3kb fragments) were purified from agarose gel as described in methods section.
Nextera XT DNA sample prep kit (Illumina FC1311024)
OTHER (in vitro strand transfer assay)

tag: band size

value: ~3 kilobases


title: HEK293 T-Rex LARP7 ko stable FH-LARP7 F44A rescue U6 snRNA RiboMeth-Seq replicates 1-2

description:
HEK293 T-Rex LARP7 ko LARP7 F44A rescue replicates 1-2
small RNA sequencing data
processed data file: RMS_LARP7_ko_rescue_U6.xlsx
rescue_F44A_rep1

source_name: HEK293 T-Rex LARP7 knockout stably expressing FH-LARP7 F44A mutant

growth_protocol: HEK293 T-Rex LARP7 ko cells stably expressing FH-LARP7 F44A mutant were cultivated in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin, 5 μg/mL blasticidin and 100 μg/mL hygromicin B. Cells were grown under standard conditions at 37°C in 5% CO2 atmosphere.


title: S4A2_R1

description:
gel excised dsDNA
dsDNA sequencing


title: S1A1_R1

description:
gel excised dsDNA
dsDNA sequencing


title: S3A2_R1

description:
gel excised dsDNA
dsDNA sequencing


title: N3


title: N1


title: N2


title: Treat_030_RBG18933

description: Treated_ICGScluster2_015_HSCs (CD31+kit+CD45-)


title: Ctrl_074_RBG19196

description: Ctrl_ICGScluster7_003_HSCs (CD31+kit+CD45-)

value: Ctrl


title: Ctrl_075_RBG19208

description: Ctrl_ICGScluster2_024_HSCs (CD31+kit+CD45-)


title: Ctrl_073_RBG19184

description: Ctrl_ICGScluster1_036_HSCs (CD31+kit+CD45-)


title: Ctrl_072_RBG19172

description: Ctrl_ICGScluster2_023_HSCs (CD31+kit+CD45-)


title: CLL10

value: CLL

value: Bone marrow buffy coat


title: Ctrl_071_RBG19160

description: Ctrl_ICGScluster1_035_HSCs (CD31+kit+CD45-)


title: CLL09


title: CLL08


title: S2A1_R1

description:
gel excised dsDNA
dsDNA sequencing


title: CLL07


title: Ctrl_070_RBG19148

description: Ctrl_ICGScluster2_022_HSCs (CD31+kit+CD45-)


title: CLL06


title: ω3LC1.4_T33_2: Liver_diet_fish8

city: St. John's

first: Xi

last: Xue

state: NL

address: 1 Marine Lab Road

department: Department of Ocean Sciences

country: Canada

institute: Memorial University of Newfoundland

hyb_protocol: Equal quantities (825 ng) of each labeled aRNA (refer to label protocol) from one experimental sample and the common reference were pooled, fragmented following the manufacturer's instructions and co-hybridized to a cGRASP-designed Agilent 44K salmonid oligonucleotide microarray (GEO accession # GPL11299) as per the manufacturer’s instructions (Agilent, Mississauga, ON). The arrays were hybridized at 65°C for 17 h with 10 rpm rotation in an Agilent hybridization oven. The array slides were washed immediately following hybridization as per the manufacturer's instructions.

scan_protocol: Each microarray was scanned at 5 µm resolution and 90% of laser power using a ScanArray Gx Plus scanner and ScanArray Express software (v4.0; Perkin Elmer, Woodbridge, ON) with photomultiplier tube (PMT) set to balance fluorescence signal between channels.

data_processing: Signal intensity data was extracted from tiff images using Imagene v9.0 (BioDiscovery). In addition to automated quality flagging, spots affected by dust particles, scratches and other local artefacts were flagged manually. Raw data was read into Bioconductor package 'marray', base 2 logratios were calculated from background-corrected median signals and control spots were removed. Data was normalized using printtip loess. Logratio data for a particular spot was removed when the raw signal value in one of the channels of that spot was lower than the threshold, which was calculated as (median background + 2 * SD) for each channel on each array. Logratio data for a particular spot was also removed if the spot was flagged in Imagene.

organism: Salmo salar

source_name: Liver tissue, ω3LC1.4 diet, individual fish

label_protocol: Eight individual fish from each experimental diet groups were used in the microarray analysis using a common reference design. An equal quantity of each DNase I-treated, column-purified liver total RNA sample involved in the microarray experiment was pooled to make a common reference for the microarray hybridizations. Anti-sense amplified RNA (aRNA) was in vitro transcribed from 1 µg of each experimental RNA or reference pooled RNA using Ambion's Amino Allyl MessageAmp II aRNA Amplification kit (Life Technologies), following the manufacturers' instructions. The quality and quantity of aRNA were assessed using UV spectrophotometry and agarose gel electrophoresis. Twenty µg of aRNA was precipitated overnight following standard molecular biology procedures and re-suspended in coupling buffer; the resulting solution was used in a labeling reaction following the manufacturer's protocol. Resulting aRNA was labeled with either Cy3 (for the common reference) or Cy5 (for the experimental individuals) fluor (GE HealthCare, Mississauga, ON) through a dye-coupling reaction, following the manufacturer's instructions. The labeling efficiency was measured using the microarray function of the NanoDrop spectrophotometer.

growth_protocol: Atlantic salmon post-smolts (average weight 178 g) were randomly distributed among 12 tanks (500 L) with flow-through seawater  (~12°C, dissolved oxygen ≥ 10 mg L-1), and all fish were kept on a photoperiod of 12 hours light / 12 hours dark. Quadruplicate tanks of fish were fed each experimental diet to apparent satiety, twice each day for a period of 14 weeks. At the end of trial, 5 fish from each tank were euthanized with 400 mg L-1 of tricaine-methane-sulfonate (TMS; Syndel Laboratories, Vancouver, BC) after 24 h of fasting. Body weight and fork length of fish were measured and recorded. Liver tissues (50-100 mg sample-1) were rapidly dissected from fish, placed in RNase-free 1.5 mL tubes, flash-frozen in liquid nitrogen, and stored at -80°C until RNA extractions were performed.

extract_protocol: Liver samples were homogenized in 1 ml of TRIzol reagent (Invitrogen/Life Technologies, Burlington, ON) with stainless steel beads (5 mm; QIAGEN, Mississauga, ON) using a TissueLyser (QIAGEN), frozen on dry ice and stored at -80°C. Frozen homogenates were slowly thawed on wet ice and the TRIzol total RNA extractions were then completed following the manufacturer’s instructions. Due to low 260/230 ratios (i.e. ranging from 0.9 to 1.7) following TRIzol extraction, all RNA samples were re-extracted using the phenol-chloroform phase separation method. Briefly, to separate the TRIzol-extracted RNA from organic materials, an equal volume of acid phenol:chloroform:isoamyl alcohol (125:24:1) (pH 4.5) (AM9720; Ambion/Life Technologies) was added to the samples, vortexed for 30 sec and centrifuged at 16,100 x g at 4°C for 20 min. The RNA was then recovered from the aqueous layer from the previous step by precipitation with 0.1 volumes of 3 M sodium acetate (pH 5.5) (Ambion/Life Technologies) and 2.5 volumes of anhydrous ethyl alcohol (Commercial Alcohols Inc., Brampton, ON) at -80°C for 1 h, followed by centrifugation at 16,100 x g at 4°C for 30 min. To wash the resulting RNA pellet, 1 ml of 75% ethanol was added and centrifuged at 16,100 x g at 4°C for 20 min. The ethanol was removed, and the RNA pellet was air-dried at room temperature for 10 min and resuspended in nuclease-free water (Invitrogen/Life Technologies). Total RNA of each sample was then treated with DNase I (QIAGEN) to degrade residual genomic DNA, and all samples were purified from salts, proteins and nucleotides using the RNeasy Mini Kit (QIAGEN) following the manufacturer’s protocols.

tag: tissue

value: Liver

tag: life stage

value: Juvenile

source_name: pool of RNAs from liver tissues invovled in the microarray experiment (i.e. FO, 100COSEFM10CM)


title: ω3LC1.4_T33_1: Liver_diet_fish7


title: ω3LC1.4_T26_2: Liver_diet_fish6


title: Ctrl_068_RBG19124

description: Ctrl_ICGScluster5_002_HSCs (CD31+kit+CD45-)


title: Treat_029_RBG19016

description: Treated_ICGScluster2_014_HSCs (CD31+kit+CD45-)


title: ω3LC1.4_T3_3: Liver_diet_fish1


title: CLL03


title: CLL02


title: Treat_028_RBG19004

description: Treated_ICGScluster1_007_HSCs (CD31+kit+CD45-)


title: ω3LC1.4_T19_2: Liver_diet_fish3


title: Ctrl_069_RBG19136

description: Ctrl_ICGScluster5_003_HSCs (CD31+kit+CD45-)


title: ω3LC1.4_T19_3: Liver_diet_fish4


title: CLL04


title: Treat_026_RBG18980

description: Treated_ICGScluster2_013_HSCs (CD31+kit+CD45-)


title: Treat_025_RBG18968

description: Treated_ICGScluster1_006_HSCs (CD31+kit+CD45-)


title: Treat_024_RBG18956

description: Treated_ICGScluster2_012_HSCs (CD31+kit+CD45-)


title: HEK293 PERK-KO

data_processing:
Illumina Casava1.8 software used for basecalling.
Sequenced reads were mapped to hg19 whole genome using tophat2 v2.0.13 with default parameters.
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using cufflinks v2.2.1.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include RPKM values

source_name: Human embryonic kidney

extract_protocol:
mRNAs were isolated from total RNA by NEBNEXT Poly(A) mRNA Magnetic Isolation Module (NEBE7490).
NEBNext mRNA Library Prep Master Mix Set for Illumina (E6110) was used with 250 ng of mRNA for the construction of sequencing libraries according to the manufacturer's protocol.

value: Human embryonic kidney

tag: cell line

tag: genotype

value: PERK-KO

tag: passage


title: ω3LC1_T34_3: Liver_diet_fish6

source_name: Liver tissue, ω3LC1 diet, individual fish