title: motor_neuron_F11_FDQ_backfill

description:
F11
counts_MN_smartseq2.csv.gz


title: motor_neuron_F16_FDQ_backfill

status: Public on Jul 29 2022

type: SRA

city: Basel

first: Patrick

last: Tschopp

address: Vesalgasse 1

department: Department of Environmental Sciences

country: Switzerland

institute: University of Basel

description:
F16
counts_MN_smartseq2.csv.gz

library_source: transcriptomic single cell

data_processing:
Raw reads were trimmed with Trimmomatic (v.0.39).
Trimmed reads were aligned with STAR (v.2.5.2) to the GRCg6a chicken genome.
Count tables were created with HTSeq (v.0.6.1).
Assembly: GRCg6a
Supplementary files format and content: Comma separated values file (.csv) . Count table created by HTSeq where genes are rows and cells are columns.

molecule: polyA RNA

organism: Gallus gallus

source_name: spinal cord

extract_protocol:
Backfilled spinal cord was dissociated with Papain (0,25 mg/ml) and individual fluorescent cells were transferred to PCR strips.
SMART-seq2

tag: celltype

value: motor neuron

tag: tissue

value: spinal cord

tag: age

value: embryonic day 9


title: motor_neuron_E9_EMR_backfill

description:
E9
counts_MN_smartseq2.csv.gz


title: motor_neuron_E8_EMR_backfill

description:
E8
counts_MN_smartseq2.csv.gz


title: motor_neuron_F10_FDQ_backfill

description:
F10
counts_MN_smartseq2.csv.gz


title: motor_neuron_F13_FDQ_backfill

description:
F13
counts_MN_smartseq2.csv.gz


title: motor_neuron_E7_EMR_backfill

description:
E7
counts_MN_smartseq2.csv.gz


title: motor_neuron_E6_EMR_backfill

description:
E6
counts_MN_smartseq2.csv.gz


title: motor_neuron_E34_EMR_backfill

description:
E34
counts_MN_smartseq2.csv.gz


title: motor_neuron_E32_EMR_backfill

description:
E32
counts_MN_smartseq2.csv.gz


title: motor_neuron_E33_EMR_backfill

description:
E33
counts_MN_smartseq2.csv.gz


title: motor_neuron_E30_EMR_backfill

description:
E30
counts_MN_smartseq2.csv.gz


title: motor_neuron_E3_EMR_backfill

description:
E3
counts_MN_smartseq2.csv.gz


title: motor_neuron_E27_EMR_backfill

description:
E27
counts_MN_smartseq2.csv.gz


title: motor_neuron_E29_EMR_backfill

description:
E29
counts_MN_smartseq2.csv.gz


title: motor_neuron_E23_EMR_backfill

description:
E23
counts_MN_smartseq2.csv.gz


title: motor_neuron_E24_EMR_backfill

description:
E24
counts_MN_smartseq2.csv.gz


title: motor_neuron_E21_EMR_backfill

description:
E21
counts_MN_smartseq2.csv.gz


title: motor_neuron_E18_EMR_backfill

description:
E18
counts_MN_smartseq2.csv.gz


title: motor_neuron_E2_EMR_backfill

description:
E2
counts_MN_smartseq2.csv.gz


title: motor_neuron_E20_EMR_backfill

description:
E20
counts_MN_smartseq2.csv.gz


title: motor_neuron_F12_FDQ_backfill

description:
F12
counts_MN_smartseq2.csv.gz


title: motor_neuron_E17_EMR_backfill

description:
E17
counts_MN_smartseq2.csv.gz


title: motor_neuron_E16_EMR_backfill

description:
E16
counts_MN_smartseq2.csv.gz


title: motor_neuron_E15_EMR_backfill

description:
E15
counts_MN_smartseq2.csv.gz


title: motor_neuron_E5_EMR_backfill

description:
E5
counts_MN_smartseq2.csv.gz


title: motor_neuron_E11_EMR_backfill

description:
E11
counts_MN_smartseq2.csv.gz


title: motor_neuron_E12_EMR_backfill

description:
E12
counts_MN_smartseq2.csv.gz


title: motor_neuron_E14_EMR_backfill

description:
E14
counts_MN_smartseq2.csv.gz


title: motor_neuron_E10_EMR_backfill

description:
E10
counts_MN_smartseq2.csv.gz


title: motor_neuron_E4_EMR_backfill

description:
E4
counts_MN_smartseq2.csv.gz


title: motor_neuron_E31_EMR_backfill

description:
E31
counts_MN_smartseq2.csv.gz


title: motor_neuron_E1_EMR_backfill

description:
E1
counts_MN_smartseq2.csv.gz


title: motor_neuron_E28_EMR_backfill

description:
E28
counts_MN_smartseq2.csv.gz


title: motor_neuron_E22_EMR_backfill

description:
E22
counts_MN_smartseq2.csv.gz


title: motor_neuron_E19_EMR_backfill

description:
E19
counts_MN_smartseq2.csv.gz


title: motor_neuron_E13_EMR_backfill

description:
E13
counts_MN_smartseq2.csv.gz


title: MiddleCerebralArtery_InternalCarotidArteryAneurysm#91

status: Public on Jul 03 2022

type: RNA

city: Showa-ku, Nagoya

first: Kinya

last: Yokoyama

state: Aichi

address: Tsurumai 65

department: Neurosurgery

country: Japan

institute: Nagoya University Hospital

hyb_protocol: A Cy3-labeled amplified cDNA sample (2 µg), prepared without the fragmentation reaction, was used for hybridization (24 hours at 65 ℃) to the oligonucleotide spotted microarray slide (Human Whole Genome, SurePrint G3 Human GE v2 8×60K Microarray; Agilent Technologies). Subsequent washing and drying were performed according to the Agilent hybridization protocol.

scan_protocol: The slide was scanned with an Agilent Model G2505A DNA microarray scanner.

data_processing: Signal intensity was quantified from the scanned image using Feature Extraction Software version 11.0.1.1 (Agilent Technologies).

molecule: total RNA

organism: Homo sapiens

source_name: Middle Crebral Artery, Internal Carotid Artery Aneurysm, Patient Number 91, replicate 1

label_protocol: Cyanine-3 (Cy3) labeled cDNA was prepared from 2ug cDNA using the SureTag DNA labeling kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer's instructions.  Dye incorporation and cDNA yield were checked with the NanoDrop One Spectrophotometer.

extract_protocol: Total RNA was extracted from each MCA specimen with an RNeasy Micro Kit (Qiagen). The purified total RNA was amplified with an Ovation PicoSL WTA System V2 kit (NuGEN Technologies, Bemmel, Netherlands)

treatment_protocol: We excised the vessel wall of the MCA following the reported procedure. Tissue samples were collected into RNAlater (Qiagen, Hilden, Germany) and stored at -80 °C.

value: Artery Wall

tag: gender

value: Female

tag: disease state

value: internal carotid artery aneurysm


title: SnrpB (CD1) mutant (het) embryo rep3

status: Public on Jun 15 2022

city: Montreal

first: Loydie

last: Jerome-Majewska

state: QC

address: 1001 Decarie Blvd EM0.2210

department: Pediatrics

country: Canada

institute: McGill University Health Centre Glen Site

description: Het 6

library_source: transcriptomic

data_processing:
Sequenced reads were trimmed for adaptor sequence, and masked for low-quality sequence using Trim_Galore.
High-quality (trimmed) sequenced reads were then mapped to mouse genome using STAR align, with default parameters.
Overall data QC was summarised with multiqc.
Raw count were extracted using featureCounts for each sample and summarised in one matrix.
Genome_build: mm10
Supplementary_files_format_and_content: Raw_gene_counts_matrix.txt: raw counts tab separated file
Supplementary_files_format_and_content: SnrpB_metadata

organism: Mus musculus

source_name: head

growth_protocol: Embryos were collected, and yolk sacs were used for genomic DNA extraction for genotyping.  The number of somites were counted under light microscope (Leica MZ6 Infinity1 stereomicroscope) at the time of dissection.  The head of the embryos were kept in RNA later at 4°C until RNA extraction. Two embryo heads of same genotype that had similar somite numbers were pooled before extracting RNA.

extract_protocol:
RNA extraction from samples was performed using Qiagen RNeasy kit following manufacturer’s protocol
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: No treatment

tag: cell type

value: CD1 (Head) cells

tag: strain

value: head

value: post natal day 9 embryo (E9.0)

tag: genotype

value: mutant (SnrpB+/-)

tag: cell line


title: SnrpB (CD1) mutant (het) embryo rep2

description: Het 5


title: MiddleCerebralArtery_Epilepsi#M116

source_name: Middle Crebral Artery, Epilepsi, Patient Number M116, replicate 1

value: Male

value: epilepsi


title: MiddleCerebralArtery_Epilepsi#M119

source_name: Middle Crebral Artery, Epilepsi, Patient Number M119, replicate 1


title: MiddleCerebralArtery_InternalCarotidArteryAneurysm#114

source_name: Middle Crebral Artery, Internal Carotid Artery Aneurysm, Patient Number 114, replicate 1


title: MiddleCerebralArtery_InternalCarotidArteryAneurysm#104

source_name: Middle Crebral Artery, Internal Carotid Artery Aneurysm, Patient Number 104, replicate 1


title: SnrpB (CD1) mutant (het) embryo rep1

description: Het 4


title: SnrpB (CD1) WT embryo at E9.0 rep3

description: WT 3

value: wild type (SnrpB +/+)


title: MiddleCerebralArtery_InternalCarotidArteryAneurysm#115

source_name: Middle Crebral Artery, Internal Carotid Artery Aneurysm, Patient Number 115, replicate 1


title: MiddleCerebralArtery_InternalCarotidArteryAneurysm#89

source_name: Middle Crebral Artery, Internal Carotid Artery Aneurysm, Patient Number 89, replicate 1


title: MiddleCerebralArtery_Epilepsi#M127

source_name: Middle Crebral Artery, Epilepsi, Patient Number M127, replicate 1


title: MiddleCerebralArtery_MoyamoyaDisease#69

source_name: Middle Crebral Artery, Moyamoya Disease, Patient Number 69, replicate 1

value: Moyamoya desease


title: MiddleCerebralArtery_InternalCarotidArteryAneurysm#106

source_name: Middle Crebral Artery, Internal Carotid Artery Aneurysm, Patient Number 106, replicate 1


title: MiddleCerebralArtery_MoyamoyaDisease#M126

source_name: Middle Crebral Artery, Moyamoya Disease, Patient Number 126, replicate 1


title: MiddleCerebralArtery_MoyamoyaDisease#63

source_name: Middle Crebral Artery, Moyamoya Disease, Patient Number 63, replicate 1


title: MiddleCerebralArtery_MoyamoyaDisease#73

source_name: Middle Crebral Artery, Moyamoya Disease, Patient Number 73, replicate 1


title: MiddleCerebralArtery_MoyamoyaDisease#M122

source_name: Middle Crebral Artery, Moyamoya Disease, Patient Number 122, replicate 1


title: MiddleCerebralArtery_MoyamoyaDisease#84

source_name: Middle Crebral Artery, Moyamoya Disease, Patient Number 84, replicate 1


title: MiddleCerebralArtery_MoyamoyaDisease#95

source_name: Middle Crebral Artery, Moyamoya Disease, Patient Number 95, replicate 1


title: SnrpB (CD1) WT embryo at E9.0 rep1

description: WT 1


title: SnrpB (CD1) WT embryo at E9.0 rep2

description: WT 2


title: T-L-3

status: Public on May 18 2022

city: Karaj

first: Abolfazl

last: Bahrami

address: Daneshgah

department: Biology

country: Iran

institute: University of Tehran

data_processing:
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to bostau8 whole genome using bowtie v0.12.2 with parameters -q -p 4 -e 100 -y -a -m 10 --best --strata
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Genome_build: bosTau8
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample ...

organism: Ovis aries

source_name: Endometrium

extract_protocol:
Tissues were removed, flash frozen on dry ice, and RNA was harvested using Trizol reagent. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: Endometrium

tag: fertility

value: low


title: T-H-1

value: high


title: Co-H-1

data_processing:
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to sheep whole genome using bowtie v0.12.2 with parameters -q -p 4 -e 100 -y -a -m 10 --best --strata
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Genome_build: ovr
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample ...

source_name: Ovary

value: Ovary


title: T-H-2


title: T-L-2


title: T-H-3


title: T-L-1


title: MiddleCerebralArtery_MoyamoyaDisease#66

source_name: Middle Crebral Artery, Moyamoya Disease, Patient Number 66, replicate 1


title: Co-L-3


title: Co-L-2


title: Co-H-3

source_name: Corpus Luteum

value: Corpus Luteum


title: Co-L-1


title: WT, LUNG

status: Public on Apr 02 2022

city: Cleveland

first: Olga

last: Cherepanova

state: OH

address: 9500 Euclid Ave

department: Cardiovascular and Metabolic Sciences

country: USA

institute: Cleveland Clinic

data_processing:
Samples were demultiplexed using cellranger mkfastq pipeline
Counts obtained using cellranger count command
Genome_build: mm10
Supplementary_files_format_and_content: csv files containing feature-sample count matrices were generated using R

source_name: Mouse lungs

extract_protocol:
Aortas, including arch, thoracic, and abdominal portions and lung left lobes from EC-Oct4Δ/Δ (n = 3) and EC-Oct4+/+ (n = 2) female mice after 5 weeks of WD feeding were cleaned, cut, and digested with all buffers and solutions contained 1 µg/mL Actinomycin D (Gibco, 11805017) to prevent transcriptional changes induced by digestion. LoBind pipet tips (Xtip Biotix, R1000-9FC) were used to transfer samples. The cell suspension was transferred to 0.04% Ultra-Pure nonacetylated BSA (Thermo Fisher, AM2616) in PBS, filtered through 35 µm filters (Falcon, 352235) on ice, and sorted on a BD Influx sorter with the 100 µm nozzle into LoBind tubes (Eppendorf, 022431048) containing PBS with 0.04% Ultra-Pure nonacetylated BSA. Cdh5 negative littermate mice after 5 weeks of WD feeding were used as a control to set the YFP+ gate.
The cells in each group were captured in Chromium 10X genomics libraries, which after barcoding, were pooled and sequenced on the Illumina MiSeq platform, 150 cycle high-output.

treatment_protocol: Animals were fed a Wester diet for 5 weeks

value: lung

value: sorted eYFP+ endothelial cells

value: Oct4 wild type

tag: treatment

value: Western diet for 5 weeks


title: KO, AORTA

source_name: Mouse aorta

treatment_protocol: Animals were fed a Western diet for 5 weeks

value: aorta

value: Oct4 knockout


title: KO, LUNG


title: MiddleCerebralArtery_MoyamoyaDisease#59

source_name: Middle Crebral Artery, Moyamoya Disease, Patient Number 59, replicate 1

value: Artery Wall

value: Female


title: Zac1_S6

status: Public on Feb 14 2022

city: RALEIGH

first: Dereje

last: Jima

state: NC

address: 850 Main Campus Drive

department: CHHE

institute: NCSU

data_processing:
The quality of sequenced data was assessed using fastqc application and 12 poor quality bases were trimmed from the 5’-end. The remaining good quality reads were aligned to the Mouse reference genome (mm38 version 87) downloaded from Ensembl database using STAR aligner. For each replicate, per-gene counts of uniquely mapped reads were calculated using htseq-count script from the HTSeq python package. The count matrix was imported to R statistical computing environment for further analysis. Initially, genes that have no count in most of the replicate sample were discarded. The remaining count data were normalized for sequencing depth and distortion, and dispersion was estimated using DESeq2 Bioconductor package in the R statistical computing environment. We fitted a leaner model using the treatment levels and a differential expressed genes were identified after applying multiple testing correction using Benjamini-Hjockberg procedure. The final significant genes were generated using padj<0.05.
Genome_build: Ensembl mm38 version 87
Supplementary_files_format_and_content: csv format  normalized count in log scale

extract_protocol:
RNA was isolated from frozen pellets using the NucleoSpin kit (Macherey Nagel) and quantified using a Nanodrop 2000. Dnase I treatment was performed as part of the protocol.
RNA-seq libraries were constructed using NEBNext Ultra Directional RNA Library Prep Kit and NEBNext Mulitplex Oligos for Illumina (NEB).

treatment_protocol: Cultured mouse hepatocytes (AML12 cells) were transfected with over-expression constructs containing either the Zac1 or EGFP open reading frames and the resulting global transcriptional changes were analyzed by RNA-seq after 24h.


title: EGFP_S3

source_name: EGFP

value: EGFP


title: EGFP_S2


title: Zac1_S5


title: Zac1_S4


title: EGFP_S1


title: MetS-Ct_97

source_name: PND21 liver

extract_protocol:
Livers were removed and flash frozen. RNA was isolated from liver samples using the NucleoSpin kit (Macherey Nagel) and quantified using a Nanodrop 2000. Dnase I treatment was performed as part of the protocol.
RNA-seq libraries were constructed using NEBNext Ultra Directional RNA Library Prep Kit and NEBNext Mulitplex Oligos for Illumina (NEB).

treatment_protocol: C57Bl/6J female F0 mice were fed a 10% or 45% fat diet (‘control’ (Ct) and ‘high fat’ (HF), respectively) from 3 weeks of age for 7 or 17 weeks, herein referred to as the 7w and 17w cohorts, respectively, and mated to males fed a control diet. On the day of birth, F1 litters were standardized to 5 pups and cross-fostered to dams who had given birth on the same day. Mice were sacrificed at postnatal day 21.

value: liver

value: postnatal day 22

value: MetS-Ct


title: MetS-Ct_85

value: postnatal day 21


title: MiddleCerebralArtery_MoyamoyaDisease#98

source_name: Middle Crebral Artery, Moyamoya Disease, Patient Number 98, replicate 1

value: Moyamoya desease


title: MetS-Ct_70


title: Co-H-2

value: high


title: Ct-MetS_65

value: Ct-MetS


title: Ct-MetS_50


title: Ct-Ct_4

value: Ct-Ct


title: MetS-Ct_53


title: Ct-MetS_63


title: MetS-MetS_157

value: MetS-MetS


title: WT, AORTA

value: sorted eYFP+ endothelial cells


title: MetS-Ct_149


title: MetS-MetS_39


title: Ct-Ct_33


title: Ct-MetS_169


title: Ct-MetS_24


title: Ct-Ct_139


title: Ct-Ct_129


title: Ct-Ct_130


title: Ct-MetS_145


title: MiddleCerebralArtery_MoyamoyaDisease#M120

source_name: Middle Crebral Artery, Moyamoya Disease, Patient Number 120, replicate 1


title: MetS-MetS_154


title: MetS-Ct_120


title: MetS-MetS_110


title: MetS-MetS_109


title: Ct-Ct_10


title: LUNG054T1My_S2

status: Public on Dec 29 2021

city: San Francisco

first: Bushra

last: Samad

state: CA

address: 505 Parnassus Ave

institute: USCF Immunoprofiler

data_processing:
RNAseq reads were first aligned to reference of rRNA and mitochondrial rRNA using BWA-mem
The remaining reads were aligned to the Ensembl GRCh38.85 transcriptome build using STAR
Genome_build: GRCh38

source_name: Tumor assocaited HLA DR+ CD11b+ CD3-CD1920-CD56- myeloid cells  sorted by flow cytometry from a primary Bladder tumor specimen

extract_protocol:
The mRNA was isolated via DinaBead Direct, then converted into amplified   cDNA using Tecan Ovation RNA-Seq System V2 kit, following the manufacturer protocol.
The dsDNA went through tagmentation, amplification and clean up with   AMPure XP beads steps, using the Illumina Nextera XT DNA Library Prep Kit. Quality control analysis was performed on the resulting pooled libraries with the Agilent Bioanalyzer HS DNA chip to assess fragment size distribution and concentration

value: Lung

tag: sorted compartment

value: myeloid

tag: disease

value: Lung cancer


title: LUNG052T1My_S17


title: LUNG052T1Li2_S40

source_name: Tumor infiltrating viable cells sorted by flow cytometry from a primary Lung tumor specimen

value: live


title: LUNG074_T1_rna_tcell_S26

source_name: Tumor assocaited conventional T cell both CD4+ and CD8+ sorted by flow cytometry from a primary Lung tumor specimen

value: tcell


title: LUNG070_T1_rna_live_S5


title: LUNG030T2_My_S26


title: IPILUNG088T1_rna_live_S24


title: LUNG049T1Li_S5


title: LUNG081T1Li_S64


title: LUNG077T1Li_S37


title: IPILUNG088T1_rna_myeloid_S6

source_name: Tumor assocaited HLA DR+ CD3-CD1920-CD56- myeloid cells  sorted by flow cytometry from a primary Bladder tumor specimen


title: LUNG030T1_My_S19


title: LUNG051T1Li2_S22


title: LUNG030T1_Li_S21


title: LUNG051T1My_S11


title: LUNG021T1_My_S35


title: LUNG074_T1_rna_live_S38


title: LUNG080T1Li_S28


title: LUNG069_T1_rna_live_S13


title: LUNG076T1Li_S1


title: LUNG015_T1_rna_live_S32


title: LUNG021T1_Li_S66


title: LUNG013_T1_rna_live_S53


title: LUNG014_T1_rna_myeloid_S4


title: LUNG013_T1_rna_myeloid_S33


title: LUNG014_T1_rna_live_S19


title: LUNG015_T1_rna_myeloid_S42


title: LUNG009_T1_rna_live_S6


title: LUNG009_T1_rna_myeloid_S39


title: LUNG008_T1_rna_myeloid_S49


title: LUNG006-T1Total10k_S64_S20_L004


title: LUNG006-T1Myeloid2k_S75_S22_L001


title: IPIKID108T1_rna_live3_S17

source_name: Tumor infiltrating viable cells sorted by flow cytometry from a primary Kidney tumor specimen

value: Kidney

value: Kidney cancer


title: IPIKID108T1_rna_myeloids_S4

source_name: Tumor assocaited HLA DR+ CD3-CD1920-CD56- myeloid cells  sorted by flow cytometry from a primary Kidney tumor specimen


title: LUNG003-T1Live2k_S70_S13_L001


title: LUNG008_T1_rna_live_S5


title: LUNG003-T1Myeloid2k_S72_S25_L001


title: IPIKID104_T1_rna_Myeloid_S2


title: IPIKID102_T1_rna_Live_S15


title: IPIKID100_T1_rna_Live_S3


title: IPIKID103_T1_rna_Live_S9


title: IPIKID103_T1_rna_Myeloid_S33


title: IPIKID104_T1_rna_Live_S10


title: IPIKID106T1_rna_live_S59


title: IPIKID109T1_rna_myeloids_S62


title: IPIKID098_T1_rna_myeloid_S14


title: IPIKID102_T1_rna_Myeloid_S21


title: IPIKID095_T1_rna_Myeloid_S28


title: IPIKID099_T1_rna_live_S26


title: KID094T1_My_S2


title: IPIKID099_T1_rna_myeloid_S5


title: KID093T1_My_S47


title: IPIKID095_T1_rna_live_S27


title: KID094T1_Li_S16


title: IPIKID100_T1_rna_Myeloid_S18


title: IPIKID098_T1_rna_live_S61


title: IPIKID097_T1_rna_live_S49


title: KID091T1_My_S39

source_name: Tumor assocaited HLA DR+ CD11b+ CD3-CD1920-CD56- myeloid cells  sorted by flow cytometry from a primary Kidney tumor specimen


title: KID092T1_My_S57


title: KID092T1_Li_S22


title: KID091T1_Li_S35


title: KID089T1_Li_S45


title: KID089T1_My_S19


title: KID084T1_Li_S26


title: KID088T1_My_S18


title: KID083T1_Li_S43


title: KID085T1_Li_S33


title: KID085T1_My_S32


title: KID092T1_Tc_S23

source_name: Tumor assocaited conventional T cell both CD4+ and CD8+ sorted by flow cytometry from a primary Kidney tumor specimen


title: KID084T1_My_S19


title: KID086T1_My_S54


title: KID088T1_Li_S38


title: KID093T1_Li_S1


title: KID079T1Li_S63


title: KID083T1_My_S23


title: KID076T1Li_S47


title: KID076T1My_S30


title: KID074_T1_rna_myeloid_S26


title: KID074_T1_rna_live_S26


title: KID086T1_Li_S47


title: KID073_T1_rna_live_S14


title: KID071_T1_rna_myeloid_S14


title: KID077T1Li_S46


title: KID078T1My_S52


title: KID071T1_Li_S38


title: KID079T1My_S31


title: KID077T1My_S46


title: KID069_T1_rna_myeloid_S1


title: KID069_T1_rna_live_S30


title: KID078T1Li_S55


title: KID062T1_My_S18


title: KID061_T1_rna_myeloid_S15


title: KID061_T1_rna_live_S24


title: KID070_T1_rna_live_S17


title: KID067T1Li_S19


title: KID066T1_Tc_S18_L001


title: KID070_T1_rna_myeloid_S6


title: KID052T1My_S53


title: KID060T1_My_S46


title: KID062_T1_rna_live_S23


title: KID067T1My_S47


title: KID057T1Li_S212


title: KID052T1Li2_S8


title: KID060_T1_rna_live_S23


title: KID051T1My_S11


title: KID051T1Li_S13


title: KID048T1Lib_S69_S10


title: KID047_T1_rna_myeloid_S30


title: KID047T1_Li_S17_L001


title: IPIHNSC078T1_rna_live_S9

source_name: Tumor infiltrating viable cells sorted by flow cytometry from a primary tumor specimen located in the head and neck region

value: Head & Neck

value: Head & Neck cancer


title: IPIHNSC078T1_rna_myeloid_S27

source_name: Tumor assocaited HLA DR+ CD3-CD1920-CD56- myeloid cells  sorted by flow cytometry from a primary tumor specimen located in the Head and Neck region


title: KID027_T1_rna_myeloid_S32


title: KID066_T1_rna_live_S46


title: KID069T1_Tc_S24


title: IPIHNSC079T1_rna_myeloid_S33


title: KID027T1_Li_S50


title: IPIHNSC079T1_rna_live_S25


title: IPIHNSC077T1_rna_live_S23


title: IPIHNSC076_T1_rna_live_S32


title: HNSC070T1_My_S48

source_name: Tumor assocaited HLA DR+ CD11b+ CD3-CD1920-CD56- myeloid cells  sorted by flow cytometry from a primary tumor specimen located in the Head and Neck region


title: HNSC070T1_Li_S12


title: IPIHNSC074_T1_rna_live_S37


title: HNSC063T1My_S31_S17


title: HNSC069T1_My_S32


title: IPIHNSC074_T1_rna_myeloid_S33


title: IPIHNSC076_T1_rna_myeloid_S11


title: HNSC063T1Li_S15_S21


title: HNSC069T1_Li_S52


title: HNSC073T1_My_S44


title: HNSC062T1Li_S83_S16


title: HNSC061T1_My_S44


title: HNSC060T1My_S32


title: HNSC061T1_Li_S27


title: HNSC060T1Li_S41


title: HNSC059T1Li_S49


title: IPIHNSC077T1_rna_myeloid_S13


title: HNSC057T1My_S52_S2


title: HNSC057T1Li_S39_S12


title: HNSC054T1Li_S54


title: HNSC031T1_My_S49


title: HNSC056_T1_rna_live_S19


title: IPIHNSC050_T1_rna_myeloid_S8


title: HNSC054T1My_S50


title: IPIHNSC050_T1_rna_live_S29


title: HNSC031T1_Li_S24


title: HNSC030T1Li_S7


title: HNSC026T1My_S72_S27


title: HNSC062T1My_S65_S32


title: HNSC026T1Li_S8_S46


title: IPIGYN124T1_rna_live_S43

source_name: Tumor infiltrating viable cells sorted by flow cytometry from a primary Gynecologic tumor specimen

value: Uterus & adnexa

value: Gynecologic cancer


title: IPIGYN110_T1_rna_live_S30


title: IPIGYN124T1_rna_myeloids_S42

source_name: Tumor assocaited HLA DR+ CD3-CD1920-CD56- myeloid cells  sorted by flow cytometry from a primary Gynecologic tumor specimen


title: HNSC021T1_Li_S10


title: IPIGYN125T1_rna_live_S57


title: IPIGYN125T1_rna_myeloids_S60


title: IPIGYN109T1_rna_live_S15


title: IPIGYN116T1_rna_myeloids_S23


title: IPIGYN116T1_rna_live_S48


title: IPIGYN108T1_rna_myeloids_S36


title: IPIGYN108T1_rna_live_S55


title: IPIGYN109T1_rna_myeloids_S28


title: IPIGYN107T1_rna_myeloid_S26


title: IPIGYN110_T1_rna_Myeloid_S13


title: IPIGYN107T1_rna_live_S21


title: IPIGYN106_T1_rna_Myeloid_S57


title: IPIGYN108_T1_rna_tcell_S20

source_name: Tumor assocaited conventional T cell both CD4+ and CD8+ sorted by flow cytometry from a primary Gynecologic tumor specimen


title: IPIGYN106_T1_rna_Live_S42


title: IPIGYN104_T1_rna_myeloid_S13


title: IPIGYN103_T1_rna_Myeloid_S44


title: IPIGYN104_T1_rna_live_S12


title: IPIGYN105_T1_rna_Myeloid_S14


title: IPIGYN100T1_rna_myeloid_S52


title: IPIGYN103_T1_rna_live_S24


title: IPIGYN105_T1_rna_Live_S25


title: IPIGYN099_T1_rna_Myeloid_S60


title: IPIGYN100_T1_rna_live_S30


title: GYN098_T1_rna_live_S40


title: GYN096T1_My_S38


title: IPIGYN099_T1_rna_Live_S52


title: GYN098_T1_rna_myeloid_S26


title: GYN093T1_My_S39

source_name: Tumor assocaited HLA DR+ CD11b+ CD3-CD1920-CD56- myeloid cells  sorted by flow cytometry from a primary Gynecologic tumor specimen


title: GYN091T1_My_S32


title: GYN093T1_Li_S35


title: GYN091T1_Li_S47


title: GYN087T1_My_S26


title: GYN089T1_My_S21


title: GYN088T1_Li_S3


title: GYN086T1_My_S14


title: GYN087T1_Li_S18


title: GYN089T1_Li_S31


title: GYN085T1_My_S30


title: GYN085T1_Li_S42


title: GYN082T1Li_S40


title: GYN082T1My_S25


title: GYN083T1Li_S32


title: IPIGYN069T1_rna_live_S40


title: GYN078T1My_S3


title: GYN078T1Li_S23


title: IPIGYN069T1_rna_myeloids_S46


title: GYN065T1Li_S208


title: IPIGYN064T1_rna_myeloid_S7


title: GYN065T1My_S226


title: IPIGYN064T1_rna_live_S14


title: GYN060T1Li_S43


title: GYN059T1My_2_S29


title: GYN096T1_Li_S67


title: GYN059T1Li_S23


title: IPIGYN048T1_rna_myeloid_S35


title: IPIGYN048T1_rna_live_S53


title: GYN060T1My_S44


title: GYN043T1My_S5


title: GYN043T1Li_S27


title: GYN013T1_Li_S36_L004


title: GYN086T1_Li_S11


title: GYN045T1My_S33


title: GYN040T1My_S38


title: GYN040T1Li_S33


title: GYN011T1_My_S10_L001


title: GYN027T1Li_S49


title: GYN045T1Li_S210


title: GYN007T1_My_S3


title: GYN007T1_Li_S30_L004


title: GYN003-T1Myeloid1k_S21_S23_L001


title: GYN011T1_Li_S18_L001


title: GYN003-T1Live10k_S19_S43_L008


title: IPICRC090T1_rna_myeloid_S34

source_name: Tumor assocaited HLA DR+ CD3-CD1920-CD56- myeloid cells  sorted by flow cytometry from a primary Colorectal tumor specimen

value: Colon & Rectum

value: Colorectal cancer


title: IPICRC093T1_rna_myeloid_S20


title: IPICRC093T1_rna_live_S11

source_name: Tumor infiltrating viable cells sorted by flow cytometry from a primary Colorectal tumor specimen


title: GYN013T1_My_S1_L001


title: IPICRC089T1_rna_myeloid_S31


title: IPICRC077_T1_rna_Myeloid_S9


title: IPICRC087T1_rna_live_S28


title: IPICRC080T1_rna_live_S38


title: IPICRC089T1_rna_live_S39


title: IPICRC077_T1_rna_Live_S22


title: IPICRC076_T1_rna_myeloid_S6


title: IPICRC076_T1_rna_live_S9


title: IPICRC090T1_rna_live_S17


title: IPICRC079_T1_rna_Live_S31


title: IPICRC075_T1_rna_live_S7


title: IPICRC074_T1_rna_live_S17


title: IPICRC073_T1_rna_myeloid_S54


title: IPICRC072_T1_rna_live_S17


title: IPICRC071_T2_rna_Live_S63

source_name: Tumor infiltrating viable cells sorted by flow cytometry from a metastastic Colorectal tumor specimen


title: IPICRC080T1_rna_myeloid_S31


title: IPICRC087T1_rna_myeloid_S37


title: CRC063T1_My_S55

source_name: Tumor assocaited HLA DR+ CD11b+ CD3-CD1920-CD56- myeloid cells  sorted by flow cytometry from a primary Colorectal tumor specimen


title: CRC067T1_My_S6


title: CRC070_T1_rna_myeloid_S16


title: CRC067T1_Li_S46


title: CRC070_T1_rna_live_S2


title: CRC060T1_Li_S65


title: CRC055T1My_S44


title: IPICRC079_T1_rna_Myeloid_S23


title: CRC064T1_My_S53


title: IPICRC062_T1_rna_live_S36


title: CRC063T1_Li_S39


title: CRC058T1_My_S49


title: CRC064T1_Li_S40


title: CRC058T1_Li_S26


title: CRC060T1_My_S52


title: CRC062T1_My_S2


title: IPICRC074_T1_rna_myeloid_S44


title: IPICRC035_T1_rna_live_S23


title: IPICRC073_T1_rna_live_S10


title: IPICRC032_T1_rna_live_S30


title: Hi-C WT

first: Terumi

last: Kohwi-Shigematsu

address: 513 Parnassus Ave (HSW810)

department: Department of Orofacial Sciences

institute: UCSF

description: This Sample includes 2 combined replicates.

library_source: genomic

data_processing:
Hi-C data processing   Hi-C datasets were analyzed using the HiC-bench platform (Lazaris, et al., 2017, BMC Genomics 18, 22, doi:10.1186/s12864-016-3387-6.).    Briefly, data were aligned against the mouse reference genome (mm10) by bowtie2 (version 2.3.1) with mostly default parameters (specific settings: --very-sensitive-local --local). For Hi-C, aligned reads were filtered by the GenomicTools (Tsirigos et al., 2012, Bioinformatics 28, 282-283) tools-hic filter command integrated in HiC-bench.  Interaction matrices for each chromosome separately were created by the HiC-bench platform at a 10kb resolution. The Hi-C filtered contact matrices were normalized using “iterative correction and eigenvector decomposition” (ICE) algorithm (Imakaev et al., 2012, Nat Methods 9, 999-1003) build into HiC-bench. To account for variances of read counts of more distant loci, distance normalization for each matrix was performed as recently described (Gong et al., 2018, Nat Commun 9, 542).  TADs were called using the algorithm developed within HiC-bench at 10kb bin resolution with the insulating window of 100kb. For visualization of Hi-C data, we generated HiC files using the Juicer.     Quality Control  The total numbers of reads per biological replicate for each genotype ranged from 200 to 230 million reads.  The percentage of reads aligned was >98% in all samples.  The percentage of accepted reads (ds-accepted-intra” and “ds-accepted-inter”) was in the range of 50% to 55.5%.  The absolute number of accepted intra-chromosomal read-pairs were ~90-100 million reads for each sample.  We analyzed data from individual samples as well as combined data from two replicates (~200 million reads) for each genotype.   Scaling plot was performed by aggregating the counts per 10kb distance bins.  Values were cpm-normalized by dividing by the total number of valid intra-chromosomal pairs.
assembly: mm10

molecule: genomic DNA

source_name: thymus, Satb1 WT

extract_protocol:
Hi-C was performed as described (Rao et al., Cell 159, 1665-1680 (2014).). Primary thymocyte were isolated  from Satb1+/+:Cd4-cre mice and were formaldehyde crosslinked at 2% at room temperature for 10 min. Two biological replica per sample were prepared for Hi-C.   Hi-C libraries was prepared using the Arima HiC+ kit (Arima Genomics) exactly following the associated protocol.  Paired-end sequencing was performed by Illumina NovaSeq 6000.
chromatin preparation for Hi-C experiment:
Hi-C was performed using Arima-HiC Kit.  Thymocytes were collected from 2 weeks old mice in two two biological replicates each from Satb1+/+:Cd4-cre (WT) and SATB1F/F:Cd4-cre (KO) mice. Briefly, 20 million cells were fixed in 2% formaldehyde for 10 min.  These cells were processed according to the protocols described in the Arma-HiC Kit, with initial digestion with restriction enzyme cocktail. Ends were filled and labeled with biotin, and subsequently ligated. After this step, DNA is sheared and biotin-labeled fragments were pull down.  The pulled down fragments were subjected to library preparation, followed by paired-end sequencing with Nova Seq.

value: thymus

value: thymocytes

tag: chromatin

value: Hi-C


title: CRC048T1Li_S9


title: CRC048T1My_S5


title: CRC039T1My_S9_S25


title: IPICRC075_T1_rna_myeloid_S36


title: CRC055T1Li_S49


title: Standard TKS407E Thymus KO 150s Satb1 Abcam 109122 (replica 2)

status: Public on Dec 28 2021

data_processing:
Sequenced reads were quality-checked using FastQC prior to downstream analysis. Unaligned reads were mapped to the mouse reference genome (NCBI37/mm9) using Bowtie2 (version 2.3.1).  Candidate binding sites were called using MACS2 (v2.2.7) using a q-value threshold of 0.01. Coverage tracks are presented in RPM (read pileups per million reads) and utilities from UCSC were used for creating browser tracks.
assembly: mmp9

source_name: SATB1 KO

extract_protocol:
For standard ChIP-seq, the protocol published by Kitagawa et al. (Nat Immunol 2017, 18, 173-183) was basically followed with minor modifications.   Thymocytes prepared from thymus of Satb1-/-  mice (P13) were cross-linked with 1% formaldehyde for 10 min at room temperature. After quenching cross-linking reaction (0.125M Gly at final), these cells were lysed in buffer (4% SDS, 50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 100 mM NaCl) with Protease inhibitor (Roche) and RNase inhibitor. Cross-linked DNA was sonicated using Branson sonifier (20% power, 10 sec on and 30 sec off for 4-6 cycles) in sonication buffer (0.5% Na-laurylsarcosine, 0.1% DOC, 1mM EDTA, 0.5mM EGTA, 10mM Tris-HCl, 100mM NaCl) with Protease inhibitor and RNase inhibitor to generate fragment size 300-1,000bp. For chromatin-immunoprecipitation, 1 µg of an antibody of choice were added to 10 ~ 20 µg of sonicated solubilized chromatin in 350 µl in dilution buffer (1% Triton X-100, 0.01% SDS, 1 mM EDTA, 15 mM Tris-HCl, 100 mM NaCl) and incubated overnight at 4°C. The chromatin fragments bound to antibodies (after incubation overnight) were captured using 40 µl of protein-A or protein-G Dynabeads (Invitrogen; pre-washed with 0.1% BSA [Fraction V, Sigma), by 4 hrs incubation time. After beads washing and DNA elution from beads, ChIP DNAs was reverse-crosslinked at 65oC overnight, followed by RNase-A treatment and proteinase K treatments. The recovered DNA was further purified with a QIAquick PCR purification kit (Qiagen), with elution in 15µl of Qiagen Elution Buffer (alternatively, Phenol-chloroform extraction and ethanol precipitation). The purified DNAs were subjected to ChIP-seq library construction.
Briefly for library construction, ChIP DNA fragments were end-repaired and ligated to indexed sequencing adapters (NEB or Rubicon library kit) and amplified with 8-11 rounds of PCR, depending on the DNA amount.  Using SPRI beads (Beckmancoulter), small fragments (<200bp) were removed from the amplified library. The size-selected library was sequenced to 50bp single-end read lengths on an Illumina HiSeq4000 (Vincent J. Coates Genomic Sequencing Laboratory, UC Berkeley).

value: Thymus

tag: antibody

tag: chip antibody vendor

value: Abcam

tag: chip antibody cat. #

tag: lot number

value: SATB1 KO

value: Standard ChIP-seq


title: Standard TKS407A Thymus KO 130s Satb1 1583D (replica 1)

value: Satb1 1583D

value: home made


title: IPICRC072_T1_rna_Myeloid_S38

tag: tissue

value: myeloid


title: Standard TKS407D Thymus KO 150s Satb1 1583D (replica 2)

tag: age


title: Urea TKS400C G4 Thymus KO Bioruptor Satb1 1583D (replica 2)

extract_protocol:
Thymocytes isolated from thymus of Satb1-/- mice (P13)  were cross-linked with 1% formaldehyde for 10 min at room temperature, followed by addition of glycine at 0.125 M for 5 min to quench cross-linking reaction. These cells were washed with 1X PBS, suspended in 1% BSA-PBS and cells were precipitated in a tube.  These cells were re-suspended in lysis buffer (4% SDS, 50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 100 mM NaCl) with Protease inhibitor (Roche) and RNase inhibitor.  The resulting cell lysate was incubated for 30 min at room temperature and loaded on top of 8 M urea followed by ultracentrifugation in a Beckman TL-100 Ultracentrifuge for 4-5 hrs at 45K rpm (181,000g, g=relative centrifugation force or RCF; TLS55 rotor).  Recovered chromatin pellet was sonicated to generate fragment size ranging 300–1,000bp using Branson sonifier (20% power, 10 sec on and 30 sec off for 4-6 cycles) in 300ml of sonication buffer [0.5% Na-laurylsarcosine, 0.1% deoxycholate (DOC), 1mM EDTA, 0.5mM EGTA, 10mM TrisHCl, 100mM NaCl] with Protease inhibitor and RNase inhibitor. Note: urea-purified crosslinked chromatin is highly vulnerable to sonication, therefore, it needs to adjust the sonication condition depending on cell type and/or the size of the precipitate DNA.  The sonicated chromatin with 1% Triton X-100 were dialyzed against dialysis buffer (10 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 5% glycerol), and dialyzed for 16 hrs at 4°C (this dialysis is optional).  The chromatin sample was centrifuged at 15,000g for 5 min at 4°C to remove a trace amount of residual insoluble components.   The test-reverse cross-linking of chromatin aliquot (5-10ml of the final volume) was done at 65°C for 2-3 hrs in buffer containing 1% SDS, 100 mM NaCl, 1 mM EDTA, 10mMTris.  Samples were further treated by 100 µg/ml RNase A at 37°C for 1 hr, 200 µg/ml proteinase K at 60°C for more than 3 hrs, followed by DNA purification using QIAquick PCR purification kit (Qiagen), shearing efficiency was examined by Bioanalyzer 2100 (Agilent Technologies Inc.). For chromatin-immunoprecipitation, 1 µg of an antibody of choice were added to 10 ~ 20 µg of sonicated solubilized chromatin in 350 ml in dilution buffer (1% Triton X-100, 0.01% SDS, 1 mM EDTA, 15 mM Tris-HCl, 100 mM NaCl) and incubated overnight with rotation at 4°C.  At least three chromatin samples were prepared for ChIP-seq from independent experiments to confirm the results. The chromatin fragments bound to antibodies (after incubation overnight) were captured using 20 µl of protein-A, protein-G or protein AG Dynabeads (Invitrogen; pre-washed with 0.1% BSA [Fraction V, Sigma), by 4 hrs incubation time at 4°C.  Dynabeads bound to chromatin fragments were washed twice with Binding buffer (10 mM Tris, pH8.0, 1 mM EDTA, 1 mM EGTA, 0.5% Triton X-100, 0.1% DOC, 100 mM NaCl), twice with LiCl buffer (100 mM Tris [pH 8.0], 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, and 0.5% DOC).  The tubes were changed and washed twice with 0.1M NaCl-TE buffer. The immunoprecipitated samples were eluted from Dynabeads by elution buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 100 mM NaCl and 1% SDS) at 65oC for 20 min.  After elution, ChIP DNAs was reverse-crosslinked at 65oC for 6 hrs, followed by RNase-A treatment at 37°C for 1 hr and proteinase K treatments for 3 hrs at 60°C. The recovered DNA was further purified with a QIAquick PCR purification kit (Qiagen), with elution in 15µl of Qiagen Elution Buffer (alternatively, Phenol-chloroform extraction and ethanol precipitation).
Briefly for library construction, ChIP DNA fragments were end-repaired and ligated to indexed sequencing adapters (NEB or Rubicon library kit) and amplified with 8-11 rounds of PCR, depending on the DNA amount.  Using SPRI beads (Beckmancoulter), small fragments (<200bp) were removed from the amplified library. The size-selected library was sequenced to 50bp single-end read lengths on an Illumina HiSeq4000 (Vincent J. Coates Genomic Sequencing Laboratory, UC Berkeley).

value: urea purified chromatin


title: Standard TKS401A G2 Thymus KO 70s input (replica 2)

extract_protocol:
For standard ChIP-seq, the protocol published by Kitagawa et al. (Nat Immunol 2017, 18, 173-183) was basically followed with minor modifications.   Thymocytes prepared from thymus of Satb1-/-  mice (P13) were cross-linked with 1% formaldehyde for 10 min at room temperature. After quenching cross-linking reaction (0.125M Gly at final), these cells were lysed in buffer (4% SDS, 50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 100 mM NaCl) with Protease inhibitor (Roche) and RNase inhibitor. Cross-linked DNA was sonicated using Branson sonifier (20% power, 10 sec on and 30 sec off for 4-6 cycles) in sonication buffer (0.5% Na-laurylsarcosine, 0.1% DOC, 1mM EDTA, 0.5mM EGTA, 10mM Tris-HCl, 100mM NaCl) with Protease inhibitor and RNase inhibitor to generate fragment size 300-1,000bp.  DNAs was reverse-crosslinked at 65oC overnight, followed by RNase-A treatment and proteinase K treatments. The recovered DNA was further purified with a QIAquick PCR purification kit (Qiagen), with elution in 15µl of Qiagen Elution Buffer (alternatively, Phenol-chloroform extraction and ethanol precipitation). The purified DNAs were subjected to sequencing library construction.
Briefly for library construction,  DNA fragments were end-repaired and ligated to indexed sequencing adapters (NEB or Rubicon library kit) and amplified with 8-11 rounds of PCR, depending on the DNA amount.  Using SPRI beads (Beckmancoulter), small fragments (<200bp) were removed from the amplified library. The size-selected library was sequenced to 50bp single-end read lengths on an Illumina HiSeq4000 (Vincent J. Coates Genomic Sequencing Laboratory, UC Berkeley).


title: Urea TKS400E G4 Thymus KO Bioruptor Satb1 Abcam 109122 (replica 2)


title: Urea TKS400A G4 Thymus KO input (replica 2)

extract_protocol:
Thymocytes isolated from thymus of Satb1-/- mice (P13) were cross-linked with 1% formaldehyde for 10 min at room temperature, followed by addition of glycine at 0.125 M for 5 min to quench cross-linking reaction. These cells were washed with 1X PBS, suspended in 1% BSA-PBS and cells were precipitated in a tube.  These cells were re-suspended in lysis buffer (4% SDS, 50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 100 mM NaCl) with Protease inhibitor (Roche) and RNase inhibitor.  The resulting cell lysate was incubated for 30 min at room temperature and loaded on top of 8 M urea followed by ultracentrifugation in a Beckman TL-100 Ultracentrifuge for 4-5 hrs at 45K rpm (181,000g, g=relative centrifugation force or RCF; TLS55 rotor).  Recovered chromatin pellet was sonicated to generate fragment size ranging 300–1,000bp using Branson sonifier (20% power, 10 sec on and 30 sec off for 4-6 cycles) in 300ml of sonication buffer [0.5% Na-laurylsarcosine, 0.1% deoxycholate (DOC), 1mM EDTA, 0.5mM EGTA, 10mM TrisHCl, 100mM NaCl] with Protease inhibitor and RNase inhibitor. Note: urea-purified crosslinked chromatin is highly vulnerable to sonication, therefore, it needs to adjust the sonication condition depending on cell type and/or the size of the precipitate DNA.  The sonicated chromatin with 1% Triton X-100 were dialyzed against dialysis buffer (10 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 5% glycerol), and dialyzed for 16 hrs at 4°C (this dialysis is optional).  The chromatin sample was centrifuged at 15,000g for 5 min at 4°C to remove a trace amount of residual insoluble components.   The test-reverse cross-linking of chromatin aliquot (5-10ml of the final volume) was done at 65°C for 2-3 hrs in buffer containing 1% SDS, 100 mM NaCl, 1 mM EDTA, 10mMTris.  Samples were further treated by 100 µg/ml RNase A at 37°C for 1 hr, 200 µg/ml proteinase K at 60°C for more than 3 hrs, followed by DNA purification using QIAquick PCR purification kit (Qiagen), and shearing efficiency was examined by Bioanalyzer 2100 (Agilent Technologies Inc.). The sonicated crosslinked chromatin before immunoprecipitation (input chromatin) was reverse-crosslinked at 65°C for 6 hrs, followed by RNase-A treatment at 37°C for 1 hr and proteinase K treatments for 3 hrs at 60°C. The recovered DNA was further purified with a QIAquick PCR purification kit (Qiagen), with elution in 15µl of Qiagen Elution Buffer (alternatively, Phenol-chloroform extraction and ethanol precipitation).  DNA isolated from input chromatin, at a comparable amount to that isolated from ChIP chromatin, was subjected to library preparation for sequencing.
Briefly for library construction, DNA fragments were end-repaired and ligated to indexed sequencing adapters (NEB or Rubicon library kit) and amplified with 8-11 rounds of PCR, depending on the DNA amount.  Using SPRI beads (Beckmancoulter), small fragments (<200bp) were removed from the amplified library. The size-selected library was sequenced to 50bp single-end read lengths on an Illumina HiSeq4000 (Vincent J. Coates Genomic Sequencing Laboratory, UC Berkeley).


title: Standard TKS402A G2 Thymus WT 70s input (replica 3)

source_name: SATB1 Wild-type

extract_protocol:
For standard ChIP-seq, the protocol published by Kitagawa et al. (Nat Immunol 2017, 18, 173-183) was basically followed with minor modifications.  Thymocytes prepared from thymus of Satb1+/+  mice (P13) were cross-linked with 1% formaldehyde for 10 min at room temperature. After quenching cross-linking reaction (0.125M Gly at final), these cells were lysed in buffer (4% SDS, 50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 100 mM NaCl) with Protease inhibitor (Roche) and RNase inhibitor. Cross-linked DNA was sonicated using Branson sonifier (20% power, 10 sec on and 30 sec off for 4-6 cycles) in sonication buffer (0.5% Na-laurylsarcosine, 0.1% DOC, 1mM EDTA, 0.5mM EGTA, 10mM Tris-HCl, 100mM NaCl) with Protease inhibitor and RNase inhibitor to generate fragment size 300-1,000bp. DNAs was reverse-crosslinked at 65oC overnight, followed by RNase-A treatment and proteinase K treatments. The recovered DNA was further purified with a QIAquick PCR purification kit (Qiagen), with elution in 15µl of Qiagen Elution Buffer (alternatively, Phenol-chloroform extraction and ethanol precipitation). The purified DNAs were subjected to sequencing library construction.
Briefly for library construction, DNA fragments were end-repaired and ligated to indexed sequencing adapters (NEB or Rubicon library kit) and amplified with 8-11 rounds of PCR, depending on the DNA amount.  Using SPRI beads (Beckmancoulter), small fragments (<200bp) were removed from the amplified library. The size-selected library was sequenced to 50bp single-end read lengths on an Illumina HiSeq4000 (Vincent J. Coates Genomic Sequencing Laboratory, UC Berkeley).

value: SATB1 Wild-type


title: Standard TKS407C Thymus KO 150s input 9replica 3)

extract_protocol:
For standard ChIP-seq, the protocol published by Kitagawa et al. (Nat Immunol 2017, 18, 173-183) was basically followed with minor modifications.   Thymocytes prepared from thymus of Satb1-/-  mice (P13) were cross-linked with 1% formaldehyde for 10 min at room temperature. After quenching cross-linking reaction (0.125M Gly at final), these cells were lysed in buffer (4% SDS, 50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 100 mM NaCl) with Protease inhibitor (Roche) and RNase inhibitor. Cross-linked DNA was sonicated using Branson sonifier (20% power, 10 sec on and 30 sec off for 4-6 cycles) in sonication buffer (0.5% Na-laurylsarcosine, 0.1% DOC, 1mM EDTA, 0.5mM EGTA, 10mM Tris-HCl, 100mM NaCl) with Protease inhibitor and RNase inhibitor to generate fragment size 300-1,000bp. DNAs was reverse-crosslinked at 65oC overnight, followed by RNase-A treatment and proteinase K treatments. The recovered DNA was further purified with a QIAquick PCR purification kit (Qiagen), with elution in 15µl of Qiagen Elution Buffer (alternatively, Phenol-chloroform extraction and ethanol precipitation). The purified DNAs were subjected to sequencing library construction.
Briefly for library construction, DNA fragments were end-repaired and ligated to indexed sequencing adapters (NEB or Rubicon library kit) and amplified with 8-11 rounds of PCR, depending on the DNA amount.  Using SPRI beads (Beckmancoulter), small fragments (<200bp) were removed from the amplified library. The size-selected library was sequenced to 50bp single-end read lengths on an Illumina HiSeq4000 (Vincent J. Coates Genomic Sequencing Laboratory, UC Berkeley).


title: CRC039T1Li_S26_S3

value: live


title: Hi-C KO

source_name: thymus, Satb1 KO

extract_protocol:
Hi-C was performed as described (Rao et al., Cell 159, 1665-1680 (2014).). Primary thymocytes were isolated from Satb1 F/F:Cd4-cre mice and were formaldehyde crosslinked at 2% at room temperature for 10 min. Two biological replica per sample were prepared for Hi-C.   Hi-C libraries was prepared using the Arima HiC+ kit (Arima Genomics) exactly following the associated protocol.  Paired-end sequencing was performed by Illumina NovaSeq 6000.
chromatin preparation for Hi-C experiment:
Hi-C was performed using Arima-HiC Kit.  Thymocytes were collected from 2 weeks old mice in two two biological replicates each from Satb1+/+:Cd4-cre (WT) and SATB1F/F:Cd4-cre (KO) mice. Briefly, 20 million cells were fixed in 2% formaldehyde for 10 min.  These cells were processed according to the protocols described in the Arma-HiC Kit, with initial digestion with restriction enzyme cocktail. Ends were filled and labeled with biotin, and subsequently ligated. After this step, DNA is sheared and biotin-labeled fragments were pull down.  The pulled down fragments were subjected to library preparation, followed by paired-end sequencing with Nova Seq.


title: Urea TKS400D G4 Thymus KO Satb1 Abcam 109122 (replica 1)


title: Standard TKS397A G4 Thymus KO input (replica 1)

extract_protocol:
For standard ChIP-seq, the protocol published by Kitagawa et al. (Nat Immunol 2017, 18, 173-183) was basically followed with minor modifications.  Thymocytes prepared from thymus of Satb1-/-  mice (P13) were cross-linked with 1% formaldehyde for 10 min at room temperature. After quenching cross-linking reaction (0.125M Gly at final), these cells were lysed in buffer (4% SDS, 50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 100 mM NaCl) with Protease inhibitor (Roche) and RNase inhibitor. Cross-linked DNA was sonicated using Branson sonifier (20% power, 10 sec on and 30 sec off for 4-6 cycles) in sonication buffer (0.5% Na-laurylsarcosine, 0.1% DOC, 1mM EDTA, 0.5mM EGTA, 10mM Tris-HCl, 100mM NaCl) with Protease inhibitor and RNase inhibitor to generate fragment size 300-1,000bp. DNAs were reverse-crosslinked at 65°C overnight, followed by RNaseA treatment and proteinaseK treatments.  The recovered DNA was further purified with a QIAquick PCR purification kit (Qiagen), with elution in 15µl of Qiagen Elution Buffer (alternatively, Phenol-chloroform extraction and ethanol precipitation). The purified DNAs were subjected to sequencing library construction.
Briefly for library construction, DNA fragments were end-repaired and ligated to indexed sequencing adapters (NEB or Rubicon library kit) and amplified with 8-11 rounds of PCR, depending on the DNA amount.  Using SPRI beads (Beckmancoulter), small fragments (<200bp) were removed from the amplified library. The size-selected library was sequenced to 50bp single-end read lengths on an Illumina HiSeq4000 (Vincent J. Coates Genomic Sequencing Laboratory, UC Berkeley).


title: Standard TKS407B Thymus KO 130s Satb1 Abcam 109122 (replica 1)


title: Standard TKS405C Thymus WT 180s input (replica 4)

extract_protocol:
For standard ChIP-seq, the protocol published by Kitagawa et al. (Nat Immunol 2017, 18, 173-183) was basically followed with minor modifications.  Thymocytes prepared from thymus of Satb1+/+  mice (P13) were cross-linked with 1% formaldehyde for 10 min at room temperature. After quenching cross-linking reaction (0.125M Gly at final), these cells were lysed in buffer (4% SDS, 50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 100 mM NaCl) with Protease inhibitor (Roche) and RNase inhibitor. Cross-linked DNA was sonicated using Branson sonifier (20% power, 10 sec on and 30 sec off for 4-6 cycles) in sonication buffer (0.5% Na-laurylsarcosine, 0.1% DOC, 1mM EDTA, 0.5mM EGTA, 10mM Tris-HCl, 100mM NaCl) with Protease inhibitor and RNase inhibitor to generate fragment size 300-1,000bp.  DNAs was reverse-crosslinked at 65oC overnight, followed by RNase-A treatment and proteinase K treatments. The recovered DNA was further purified with a QIAquick PCR purification kit (Qiagen), with elution in 15µl of Qiagen Elution Buffer (alternatively, Phenol-chloroform extraction and ethanol precipitation). The purified DNAs were subjected to sequencing library construction.
Briefly for library construction, DNA fragments were end-repaired and ligated to indexed sequencing adapters (NEB or Rubicon library kit) and amplified with 8-11 rounds of PCR, depending on the DNA amount.  Using SPRI beads (Beckmancoulter), small fragments (<200bp) were removed from the amplified library. The size-selected library was sequenced to 50bp single-end read lengths on an Illumina HiSeq4000 (Vincent J. Coates Genomic Sequencing Laboratory, UC Berkeley).


title: Urea TKS355A P13 Thymus WT input (replica 1)

extract_protocol:
Thymocytes isolated from thymus of Satb1+/+ mice (P13) were cross-linked with 1% formaldehyde for 10 min at room temperature, followed by addition of glycine at 0.125 M for 5 min to quench cross-linking reaction. These cells were washed with 1X PBS, suspended in 1% BSA-PBS and cells were precipitated in a tube.  These cells were re-suspended in lysis buffer (4% SDS, 50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 100 mM NaCl) with Protease inhibitor (Roche) and RNase inhibitor.  The resulting cell lysate was incubated for 30 min at room temperature and loaded on top of 8 M urea followed by ultracentrifugation in a Beckman TL-100 Ultracentrifuge for 4-5 hrs at 45K rpm (181,000g, g=relative centrifugation force or RCF; TLS55 rotor).  Recovered chromatin pellet was sonicated to generate fragment size ranging 300–1,000bp using Branson sonifier (20% power, 10 sec on and 30 sec off for 4-6 cycles) in 300ml of sonication buffer [0.5% Na-laurylsarcosine, 0.1% deoxycholate (DOC), 1mM EDTA, 0.5mM EGTA, 10mM TrisHCl, 100mM NaCl] with Protease inhibitor and RNase inhibitor. Note: urea-purified crosslinked chromatin is highly vulnerable to sonication, therefore, it needs to adjust the sonication condition depending on cell type and/or the size of the precipitate DNA.  The sonicated chromatin with 1% Triton X-100 were dialyzed against dialysis buffer (10 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 5% glycerol), and dialyzed for 16 hrs at 4°C (this dialysis is optional).  The chromatin sample was centrifuged at 15,000g for 5 min at 4°C to remove a trace amount of residual insoluble components. The test-reverse cross-linking of chromatin aliquot (5-10ml of the final volume) was done at 65°C for 2-3 hrs in buffer containing 1% SDS, 100 mM NaCl, 1 mM EDTA, 10mMTris.  Samples were further treated by 100 µg/ml RNase A at 37°C for 1 hr, 200 µg/ml proteinase K at 60°C for more than 3 hrs, followed by DNA purification using QIAquick PCR purification kit (Qiagen), and shearing efficiency was examined by Bioanalyzer 2100 (Agilent Technologies Inc.). The sonicated crosslinked chromatin before immunoprecipitation (input chromatin) was reverse-crosslinked at 65°C for 6 hrs, followed by RNase-A treatment at 37°C for 1 hr and proteinase K treatments for 3 hrs at 60°C. The recovered DNA was further purified with a QIAquick PCR purification kit (Qiagen), with elution in 15µl of Qiagen Elution Buffer (alternatively, Phenol-chloroform extraction and ethanol precipitation).  DNA isolated from input chromatin, at a comparable amount to that isolated from ChIP chromatin, was subjected to library preparation for sequencing.
Briefly for library construction, DNA fragments were end-repaired and ligated to indexed sequencing adapters (NEB or Rubicon library kit) and amplified with 8-11 rounds of PCR, depending on the DNA amount.  Using SPRI beads (Beckmancoulter), small fragments (<200bp) were removed from the amplified library. The size-selected library was sequenced to 50bp single-end read lengths on an Illumina HiSeq4000 (Vincent J. Coates Genomic Sequencing Laboratory, UC Berkeley).


title: Urea TKS356A P13 Thymus KO-input (replica 1)

extract_protocol:
Thymocytes isolated from thymus of Satb1-/- mice (P13) were cross-linked with 1% formaldehyde for 10 min at room temperature, followed by addition of glycine at 0.125 M for 5 min to quench cross-linking reaction. These cells were washed with 1X PBS, suspended in 1% BSA-PBS and cells were precipitated in a tube.  These cells were re-suspended in lysis buffer (4% SDS, 50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 100 mM NaCl) with Protease inhibitor (Roche) and RNase inhibitor.  The resulting cell lysate was incubated for 30 min at room temperature and loaded on top of 8 M urea followed by ultracentrifugation in a Beckman TL-100 Ultracentrifuge for 4-5 hrs at 45K rpm (181,000g, g=relative centrifugation force or RCF; TLS55 rotor).  Recovered chromatin pellet was sonicated to generate fragment size ranging 300–1,000bp using Branson sonifier (20% power, 10 sec on and 30 sec off for 4-6 cycles) in 300ml of sonication buffer [0.5% Na-laurylsarcosine, 0.1% deoxycholate (DOC), 1mM EDTA, 0.5mM EGTA, 10mM TrisHCl, 100mM NaCl] with Protease inhibitor and RNase inhibitor. Note: urea-purified crosslinked chromatin is highly vulnerable to sonication, therefore, it needs to adjust the sonication condition depending on cell type and/or the size of the precipitate DNA.  The sonicated chromatin with 1% Triton X-100 were dialyzed against dialysis buffer (10 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 5% glycerol), and dialyzed for 16 hrs at 4°C (this dialysis is optional).  The chromatin sample was centrifuged at 15,000g for 5 min at 4°C to remove a trace amount of residual insoluble components.   The test-reverse cross-linking of chromatin aliquot (5-10ml of the final volume) was done at 65°C for 2-3 hrs in buffer containing 1% SDS, 100 mM NaCl, 1 mM EDTA, 10mMTris.  Samples were further treated by 100 µg/ml RNase A at 37°C for 1 hr, 200 µg/ml proteinase K at 60°C for more than 3 hrs, followed by DNA purification using QIAquick PCR purification kit (Qiagen), and and shearing efficiency was examined by Bioanalyzer 2100 (Agilent Technologies Inc.). The sonicated crosslinked chromatin before immunoprecipitation (input chromatin) was reverse-crosslinked at 65°C for 6 hrs, followed by RNase-A treatment at 37°C for 1 hr and proteinase K treatments for 3 hrs at 60°C. The recovered DNA was further purified with a QIAquick PCR purification kit (Qiagen), with elution in 15µl of Qiagen Elution Buffer (alternatively, Phenol-chloroform extraction and ethanol precipitation).  DNA isolated from input chromatin, at a comparable amount to that isolated from ChIP chromatin, was subjected to library preparation for sequencing.
Briefly for library construction, DNA fragments were end-repaired and ligated to indexed sequencing adapters (NEB or Rubicon library kit) and amplified with 8-11 rounds of PCR, depending on the DNA amount.  Using SPRI beads (Beckmancoulter), small fragments (<200bp) were removed from the amplified library. The size-selected library was sequenced to 50bp single-end read lengths on an Illumina HiSeq4000 (Vincent J. Coates Genomic Sequencing Laboratory, UC Berkeley).


title: TKS120 Thymus sonic in vitro Satb1 protein low (BUR reference map)

data_processing:
Sequenced reads were quality-checked using FastQC prior to downstream analysis. Unaligned reads were mapped to the mouse reference genome (NCBI37/mm9) using Bowtie2 (version 2.3.1).  Candidate binding sites were called using MACS2 (v2.2.7) using a q-value threshold of 0.01. Coverage tracks are presented in RPM (read pileups per million reads) and utilities from UCSC were used for creating browser tracks.
assembly: mm9

source_name: in vitro

extract_protocol:
The procedure is described in Kohwi-Shigematsu, et al.,2012 (Methods, 58, 243-254).  Briefly, pure genomic DNA isolated from mouse thymocytes was sonicated with BioRuptor (UCD-200, Diagenode) to generate DNA fragments between 300 and 600 bp.  The resulting DNA fragments were allowed to bind to his-tagged Satb1recombinant protein. His-Satb1-DNA complexes are captured by incubation with the pre-blocked MagneHis beads for 5 min at 25oC. The beads were  then washed three times with Binding Buffer. His-Satb1-DNA complexes are eluted from the beads, and the DNA fragments were purified.
Briefly for library construction, ChIP DNA fragments were end-repaired and ligated to indexed sequencing adapters (NEB or Rubicon library kit).  Using SPRI beads (Beckmancoulter), small fragments (<200bp) were removed from the amplified library. The size-selected library was sequenced to 50bp single-end read lengths on an Illumina HiSeq4000 (Vincent J. Coates Genomic Sequencing Laboratory, UC Berkeley).

value: in vitro

value: in vitro SATB1-bound DNA fragments


title: Urea TKS340 6w Thymus WT input (replica 2)

extract_protocol:
Thymocytes isolated from thymus of Satb1+/+ mice (P13) were cross-linked with 1% formaldehyde for 10 min at room temperature, followed by addition of glycine at 0.125 M for 5 min to quench cross-linking reaction. These cells were washed with 1X PBS, suspended in 1% BSA-PBS and cells were precipitated in a tube.  These cells were re-suspended in lysis buffer (4% SDS, 50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 100 mM NaCl) with Protease inhibitor (Roche) and RNase inhibitor.  The resulting cell lysate was incubated for 30 min at room temperature and loaded on top of 8 M urea followed by ultracentrifugation in a Beckman TL-100 Ultracentrifuge for 4-5 hrs at 45K rpm (181,000g, g=relative centrifugation force or RCF; TLS55 rotor).  Recovered chromatin pellet was sonicated to generate fragment size ranging 300–1,000bp using Branson sonifier (20% power, 10 sec on and 30 sec off for 4-6 cycles) in 300ml of sonication buffer [0.5% Na-laurylsarcosine, 0.1% deoxycholate (DOC), 1mM EDTA, 0.5mM EGTA, 10mM TrisHCl, 100mM NaCl] with Protease inhibitor and RNase inhibitor. Note: urea-purified crosslinked chromatin is highly vulnerable to sonication, therefore, it needs to adjust the sonication condition depending on cell type and/or the size of the precipitate DNA.  The sonicated chromatin with 1% Triton X-100 were dialyzed against dialysis buffer (10 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 5% glycerol), and dialyzed for 16 hrs at 4°C (this dialysis is optional).  The chromatin sample was centrifuged at 15,000g for 5 min at 4°C to remove a trace amount of residual insoluble components.   The test-reverse cross-linking of chromatin aliquot (5-10ml of the final volume) was done at 65°C for 2-3 hrs in buffer containing 1% SDS, 100 mM NaCl, 1 mM EDTA, 10mMTris.  Samples were further treated by 100 µg/ml RNase A at 37°C for 1 hr, 200 µg/ml proteinase K at 60°C for more than 3 hrs, followed by DNA purification using QIAquick PCR purification kit (Qiagen), and shearing efficiency was examined by Bioanalyzer 2100 (Agilent Technologies Inc.). The sonicated crosslinked chromatin before immunoprecipitation (input chromatin) was reverse-crosslinked at 65oC for 4hrs, followed by RNase-A treatment at 37°C for 1 hr and proteinase K treatments for 3 hrs at 60°C. The recovered DNA was further purified with a QIAquick PCR purification kit (Qiagen), with elution in 15µl of Qiagen Elution Buffer (alternatively, Phenol-chloroform extraction and ethanol precipitation).  DNA isolated from input chromatin, at a comparable amount to that isolated from ChIP chromatin, was subjected to library preparation for sequencing.
Briefly for library construction, DNA fragments were end-repaired and ligated to indexed sequencing adapters (NEB or Rubicon library kit) and amplified with 8-11 rounds of PCR, depending on the DNA amount.  Using SPRI beads (Beckmancoulter), small fragments (<200bp) were removed from the amplified library. The size-selected library was sequenced to 50bp single-end read lengths on an Illumina HiSeq4000 (Vincent J. Coates Genomic Sequencing Laboratory, UC Berkeley).


title: Standard TKS408B wt Thymus 150s Satb1 (replica 2)

extract_protocol:
For standard ChIP-seq, the protocol published by Kitagawa et al. (Nat Immunol 2017, 18, 173-183) was basically followed with minor modifications.  Thymocytes prepared from thymus of Satb1+/+  mice (P13) were cross-linked with 1% formaldehyde for 10 min at room temperature. After quenching cross-linking reaction (0.125M Gly at final), these cells were lysed in buffer (4% SDS, 50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 100 mM NaCl) with Protease inhibitor (Roche) and RNase inhibitor. Cross-linked DNA was sonicated using Branson sonifier (20% power, 10 sec on and 30 sec off for 4-6 cycles) in sonication buffer (0.5% Na-laurylsarcosine, 0.1% DOC, 1mM EDTA, 0.5mM EGTA, 10mM Tris-HCl, 100mM NaCl) with Protease inhibitor and RNase inhibitor to generate fragment size 300-1,000bp. For chromatin-immunoprecipitation, 1 µg of an antibody of choice were added to 10 ~ 20 µg of sonicated solubilized chromatin in 350 µl in dilution buffer (1% Triton X-100, 0.01% SDS, 1 mM EDTA, 15 mM Tris-HCl, 100 mM NaCl) and incubated overnight at 4°C. The chromatin fragments bound to antibodies (after incubation overnight) were captured using 40 µl of protein-A or protein-G Dynabeads (Invitrogen; pre-washed with 0.1% BSA [Fraction V, Sigma), by 4 hrs incubation time. After beads washing and DNA elution from beads, ChIP DNAs was reverse-crosslinked at 65oC overnight, followed by RNase-A treatment and proteinase K treatments. The recovered DNA was further purified with a QIAquick PCR purification kit (Qiagen), with elution in 15µl of Qiagen Elution Buffer (alternatively, Phenol-chloroform extraction and ethanol precipitation). The purified DNAs were subjected to ChIP-seq library construction.
Briefly for library construction, ChIP DNA fragments were end-repaired and ligated to indexed sequencing adapters (NEB or Rubicon library kit) and amplified with 8-11 rounds of PCR, depending on the DNA amount.  Using SPRI beads (Beckmancoulter), small fragments (<200bp) were removed from the amplified library. The size-selected library was sequenced to 50bp single-end read lengths on an Illumina HiSeq4000 (Vincent J. Coates Genomic Sequencing Laboratory, UC Berkeley).


title: Standard TKS328 6w Thymus15s Satb1 (replica 1)


title: Urea TKS400B G4 Thymus KO Satb1 1583D (replica 1)


title: Urea TKS404E Skin 60s Satb11583D (replica 2)

extract_protocol:
Primary epidermal keratinocyotes  isolated from skin of Satb1+/+  mice (P5) was  cross-linked with 1% formaldehyde for 10 min at room temperature, followed by addition of glycine at 0.125 M for 5 min to quench cross-linking reaction. These cells were washed with 1X PBS, suspended in 1% BSA-PBS and cells were precipitated in a tube.  These cells were re-suspended in lysis buffer (4% SDS, 50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 100 mM NaCl) with Protease inhibitor (Roche) and RNase inhibitor.  The resulting cell lysate was incubated for 30 min at room temperature and loaded on top of 8 M urea followed by ultracentrifugation in a Beckman TL-100 Ultracentrifuge for 4-5 hrs at 45K rpm (181,000g, g=relative centrifugation force or RCF; TLS55 rotor).  Recovered chromatin pellet was sonicated to generate fragment size ranging 300–1,000bp using Branson sonifier (20% power, 10 sec on and 30 sec off for 4-6 cycles) in 300ml of sonication buffer [0.5% Na-laurylsarcosine, 0.1% deoxycholate (DOC), 1mM EDTA, 0.5mM EGTA, 10mM TrisHCl, 100mM NaCl] with Protease inhibitor and RNase inhibitor. Note: urea-purified crosslinked chromatin is highly vulnerable to sonication, therefore, it needs to adjust the sonication condition depending on cell type and/or the size of the precipitate DNA.  The sonicated chromatin with 1% Triton X-100 were dialyzed against dialysis buffer (10 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 5% glycerol), and dialyzed for 16 hrs at 4°C (this dialysis is optional).  The chromatin sample was centrifuged at 15,000g for 5 min at 4°C to remove a trace amount of residual insoluble components.  The test-reverse cross-linking of chromatin aliquot (5-10ml of the final volume) was done at 65°C for 2-3 hrs in buffer containing 1% SDS, 100 mM NaCl, 1 mM EDTA, 10mMTris.  Samples were further treated by 100 µg/ml RNase A at 37°C for 1 hr, 200 µg/ml proteinase K at 60°C for more than 3 hrs, followed by DNA purification using QIAquick PCR purification kit (Qiagen), shearing efficiency was examined by Bioanalyzer 2100 (Agilent Technologies Inc.). For chromatin-immunoprecipitation, 1 µg of an antibody of choice were added to 10 ~ 20 µg of sonicated solubilized chromatin in 350 ml in dilution buffer (1% Triton X-100, 0.01% SDS, 1 mM EDTA, 15 mM Tris-HCl, 100 mM NaCl) and incubated overnight with rotation at 4°C.  At least three chromatin samples were prepared for ChIP-seq from independent experiments to confirm the results. The chromatin fragments bound to antibodies (after incubation overnight) were captured using 20 µl of protein-A, protein-G or protein AG Dynabeads (Invitrogen; pre-washed with 0.1% BSA [Fraction V, Sigma), by 4 hrs incubation time at 4°C.  Dynabeads bound to chromatin fragments were washed twice with Binding buffer (10 mM Tris, pH8.0, 1 mM EDTA, 1 mM EGTA, 0.5% Triton X-100, 0.1% DOC, 100 mM NaCl), twice with LiCl buffer (100 mM Tris [pH 8.0], 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, and 0.5% DOC).  The tubes were changed and washed twice with 0.1M NaCl-TE buffer. The immunoprecipitated samples were eluted from Dynabeads by elution buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 100 mM NaCl and 1% SDS) at 65oC for 20 min.  After elution, ChIP DNAs was reverse-crosslinked at 65oC for 6 hrs, followed by RNase-A treatment at 37°C for 1 hr and proteinase K treatments for 3 hrs at 60°C. The recovered DNA was further purified with a QIAquick PCR purification kit (Qiagen), with elution in 15µl of Qiagen Elution Buffer (alternatively, Phenol-chloroform extraction and ethanol precipitation).
Briefly for library construction, ChIP DNA fragments were end-repaired and ligated to indexed sequencing adapters (NEB or Rubicon library kit) and amplified with 8-11 rounds of PCR, depending on the DNA amount.  Using SPRI beads (Beckmancoulter), small fragments (<200bp) were removed from the amplified library. The size-selected library was sequenced to 50bp single-end read lengths on an Illumina HiSeq4000 (Vincent J. Coates Genomic Sequencing Laboratory, UC Berkeley).

value: Skin


title: Urea TKS355C P13 Thymus wt Satb1 1583D 4u (replica 1)

extract_protocol:
Thymocytes isolated from thymus of Satb1+/+ mice (P13)  were cross-linked with 1% formaldehyde for 10 min at room temperature, followed by addition of glycine at 0.125 M for 5 min to quench cross-linking reaction. These cells were washed with 1X PBS, suspended in 1% BSA-PBS and cells were precipitated in a tube.  These cells were re-suspended in lysis buffer (4% SDS, 50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 100 mM NaCl) with Protease inhibitor (Roche) and RNase inhibitor.  The resulting cell lysate was incubated for 30 min at room temperature and loaded on top of 8 M urea followed by ultracentrifugation in a Beckman TL-100 Ultracentrifuge for 4-5 hrs at 45K rpm (181,000g, g=relative centrifugation force or RCF; TLS55 rotor).  Recovered chromatin pellet was sonicated to generate fragment size ranging 300–1,000bp using Branson sonifier (20% power, 10 sec on and 30 sec off for 4-6 cycles) in 300ml of sonication buffer [0.5% Na-laurylsarcosine, 0.1% deoxycholate (DOC), 1mM EDTA, 0.5mM EGTA, 10mM TrisHCl, 100mM NaCl] with Protease inhibitor and RNase inhibitor. Note: urea-purified crosslinked chromatin is highly vulnerable to sonication, therefore, it needs to adjust the sonication condition depending on cell type and/or the size of the precipitate DNA.  The sonicated chromatin with 1% Triton X-100 were dialyzed against dialysis buffer (10 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 5% glycerol), and dialyzed for 16 hrs at 4°C (this dialysis is optional).  The chromatin sample was centrifuged at 15,000g for 5 min at 4°C to remove a trace amount of residual insoluble components.   The test-reverse cross-linking of chromatin aliquot (5-10ml of the final volume) was done at 65°C for 2-3 hrs in buffer containing 1% SDS, 100 mM NaCl, 1 mM EDTA, 10mMTris.  Samples were further treated by 100 µg/ml RNase A at 37°C for 1 hr, 200 µg/ml proteinase K at 60°C for more than 3 hrs, followed by DNA purification using QIAquick PCR purification kit (Qiagen), shearing efficiency was examined by Bioanalyzer 2100 (Agilent Technologies Inc.). For chromatin-immunoprecipitation, 1 µg of an antibody of choice were added to 10 ~ 20 µg of sonicated solubilized chromatin in 350 ml in dilution buffer (1% Triton X-100, 0.01% SDS, 1 mM EDTA, 15 mM Tris-HCl, 100 mM NaCl) and incubated overnight with rotation at 4°C.  At least three chromatin samples were prepared for ChIP-seq from independent experiments to confirm the results. The chromatin fragments bound to antibodies (after incubation overnight) were captured using 20 µl of protein-A, protein-G or protein AG Dynabeads (Invitrogen; pre-washed with 0.1% BSA [Fraction V, Sigma), by 4 hrs incubation time at 4°C.  Dynabeads bound to chromatin fragments were washed twice with Binding buffer (10 mM Tris, pH8.0, 1 mM EDTA, 1 mM EGTA, 0.5% Triton X-100, 0.1% DOC, 100 mM NaCl), twice with LiCl buffer (100 mM Tris [pH 8.0], 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, and 0.5% DOC).  The tubes were changed and washed twice with 0.1M NaCl-TE buffer. The immunoprecipitated samples were eluted from Dynabeads by elution buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 100 mM NaCl and 1% SDS) at 65oC for 20 min.  After elution, ChIP DNAs was reverse-crosslinked at 65oC for 6 hrs, followed by RNase-A treatment at 37°C for 1 hr and proteinase K treatments for 3 hrs at 60°C. The recovered DNA was further purified with a QIAquick PCR purification kit (Qiagen), with elution in 15µl of Qiagen Elution Buffer (alternatively, Phenol-chloroform extraction and ethanol precipitation).
Briefly for library construction, ChIP DNA fragments were end-repaired and ligated to indexed sequencing adapters (NEB or Rubicon library kit) and amplified with 8-11 rounds of PCR, depending on the DNA amount.  Using SPRI beads (Beckmancoulter), small fragments (<200bp) were removed from the amplified library. The size-selected library was sequenced to 50bp single-end read lengths on an Illumina HiSeq4000 (Vincent J. Coates Genomic Sequencing Laboratory, UC Berkeley).


title: Urea TKS404B Skin 55s Satb11583D (replica 1)

extract_protocol:
Primary epidermal keratinocyotes  isolated from skin of Satb1+/+  mice (P5) were cross-linked with 1% formaldehyde for 10 min at room temperature, followed by addition of glycine at 0.125 M for 5 min to quench cross-linking reaction. These cells were washed with 1X PBS, suspended in 1% BSA-PBS and cells were precipitated in a tube.  These cells were re-suspended in lysis buffer (4% SDS, 50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 100 mM NaCl) with Protease inhibitor (Roche) and RNase inhibitor.  The resulting cell lysate was incubated for 30 min at room temperature and loaded on top of 8 M urea followed by ultracentrifugation in a Beckman TL-100 Ultracentrifuge for 4-5 hrs at 45K rpm (181,000g, g=relative centrifugation force or RCF; TLS55 rotor).  Recovered chromatin pellet was sonicated to generate fragment size ranging 300–1,000bp using Branson sonifier (20% power, 10 sec on and 30 sec off for 4-6 cycles) in 300ml of sonication buffer [0.5% Na-laurylsarcosine, 0.1% deoxycholate (DOC), 1mM EDTA, 0.5mM EGTA, 10mM TrisHCl, 100mM NaCl] with Protease inhibitor and RNase inhibitor. Note: urea-purified crosslinked chromatin is highly vulnerable to sonication, therefore, it needs to adjust the sonication condition depending on cell type and/or the size of the precipitate DNA.  The sonicated chromatin with 1% Triton X-100 were dialyzed against dialysis buffer (10 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 5% glycerol), and dialyzed for 16 hrs at 4°C (this dialysis is optional).  The chromatin sample was centrifuged at 15,000g for 5 min at 4°C to remove a trace amount of residual insoluble components.  The test-reverse cross-linking of chromatin aliquot (5-10ml of the final volume) was done at 65°C for 2-3 hrs in buffer containing 1% SDS, 100 mM NaCl, 1 mM EDTA, 10mMTris.  Samples were further treated by 100 µg/ml RNase A at 37°C for 1 hr, 200 µg/ml proteinase K at 60°C for more than 3 hrs, followed by DNA purification using QIAquick PCR purification kit (Qiagen), shearing efficiency was examined by Bioanalyzer 2100 (Agilent Technologies Inc.). For chromatin-immunoprecipitation, 1 µg of an antibody of choice were added to 10 ~ 20 µg of sonicated solubilized chromatin in 350 ml in dilution buffer (1% Triton X-100, 0.01% SDS, 1 mM EDTA, 15 mM Tris-HCl, 100 mM NaCl) and incubated overnight with rotation at 4°C.  At least three chromatin samples were prepared for ChIP-seq from independent experiments to confirm the results. The chromatin fragments bound to antibodies (after incubation overnight) were captured using 20 µl of protein-A, protein-G or protein AG Dynabeads (Invitrogen; pre-washed with 0.1% BSA [Fraction V, Sigma), by 4 hrs incubation time at 4°C.  Dynabeads bound to chromatin fragments were washed twice with Binding buffer (10 mM Tris, pH8.0, 1 mM EDTA, 1 mM EGTA, 0.5% Triton X-100, 0.1% DOC, 100 mM NaCl), twice with LiCl buffer (100 mM Tris [pH 8.0], 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, and 0.5% DOC).  The tubes were changed and washed twice with 0.1M NaCl-TE buffer. The immunoprecipitated samples were eluted from Dynabeads by elution buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 100 mM NaCl and 1% SDS) at 65oC for 20 min.  After elution, ChIP DNAs was reverse-crosslinked at 65oC for 6 hrs, followed by RNase-A treatment at 37°C for 1 hr and proteinase K treatments for 3 hrs at 60°C. The recovered DNA was further purified with a QIAquick PCR purification kit (Qiagen), with elution in 15µl of Qiagen Elution Buffer (alternatively, Phenol-chloroform extraction and ethanol precipitation).
Briefly for library construction, ChIP DNA fragments were end-repaired and ligated to indexed sequencing adapters (NEB or Rubicon library kit) and amplified with 8-11 rounds of PCR, depending on the DNA amount.  Using SPRI beads (Beckmancoulter), small fragments (<200bp) were removed from the amplified library. The size-selected library was sequenced to 50bp single-end read lengths on an Illumina HiSeq4000 (Vincent J. Coates Genomic Sequencing Laboratory, UC Berkeley).


title: Urea TKS346D 6w Thymus CTCF 4ul (replica 1)

value: CTCF

value: Active MOTIF


title: Urea TKS345 Brain 12 cycles Satb1 1583D (replica 2)

extract_protocol:
Single cell suspension was prepared from cortex of Satb1+/+ mice (P13).  Isolated cells (note: only neurons express SATB1) were cross-linked with 1% formaldehyde for 10 min at room temperature, followed by addition of glycine at 0.125 M for 5 min to quench cross-linking reaction. These cells were washed with 1X PBS, suspended in 1% BSA-PBS and cells were precipitated in a tube.  These cells were re-suspended in lysis buffer (4% SDS, 50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 100 mM NaCl) with Protease inhibitor (Roche) and RNase inhibitor.  The resulting cell lysate was incubated for 30 min at room temperature and loaded on top of 8 M urea followed by ultracentrifugation in a Beckman TL-100 Ultracentrifuge for 4-5 hrs at 45K rpm (181,000g, g=relative centrifugation force or RCF; TLS55 rotor).  Recovered chromatin pellet was sonicated to generate fragment size ranging 300–1,000bp using Branson sonifier (20% power, 10 sec on and 30 sec off for 4-6 cycles) in 300ml of sonication buffer [0.5% Na-laurylsarcosine, 0.1% deoxycholate (DOC), 1mM EDTA, 0.5mM EGTA, 10mM TrisHCl, 100mM NaCl] with Protease inhibitor and RNase inhibitor. Note: urea-purified crosslinked chromatin is highly vulnerable to sonication, therefore, it needs to adjust the sonication condition depending on cell type and/or the size of the precipitate DNA.  The sonicated chromatin with 1% Triton X-100 were dialyzed against dialysis buffer (10 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 5% glycerol), and dialyzed for 16 hrs at 4°C (this dialysis is optional).  The chromatin sample was centrifuged at 15,000g for 5 min at 4°C to remove a trace amount of residual insoluble components.  The test-reverse cross-linking of chromatin aliquot (5-10ml of the final volume) was done at 65°C for 2-3 hrs in buffer containing 1% SDS, 100 mM NaCl, 1 mM EDTA, 10mMTris.  Samples were further treated by 100 µg/ml RNase A at 37°C for 1 hr, 200 µg/ml proteinase K at 60°C for more than 3 hrs, followed by DNA purification using QIAquick PCR purification kit (Qiagen), and shearing efficiency was examined by Bioanalyzer 2100 (Agilent Technologies Inc.). For chromatin-immunoprecipitation, 1 µg of an antibody of choice were added to 10 ~ 20 µg of sonicated solubilized chromatin in 350 ml in dilution buffer (1% Triton X-100, 0.01% SDS, 1 mM EDTA, 15 mM Tris-HCl, 100 mM NaCl) and incubated overnight with rotation at 4°C.  At least three chromatin samples were prepared for ChIP-seq from independent experiments to confirm the results. The chromatin fragments bound to antibodies (after incubation overnight) were captured using 20 µl of protein-A, protein-G or protein AG Dynabeads (Invitrogen; pre-washed with 0.1% BSA [Fraction V, Sigma), by 4 hrs incubation time at 4°C.  Dynabeads bound to chromatin fragments were washed twice with Binding buffer (10 mM Tris, pH8.0, 1 mM EDTA, 1 mM EGTA, 0.5% Triton X-100, 0.1% DOC, 100 mM NaCl), twice with LiCl buffer (100 mM Tris [pH 8.0], 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, and 0.5% DOC).  The tubes were changed and washed twice with 0.1M NaCl-TE buffer. The immunoprecipitated samples were eluted from Dynabeads by elution buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 100 mM NaCl and 1% SDS) at 65oC for 20 min.  After elution, ChIP DNAs was reverse-crosslinked at 65oC for 6 hrs, followed by RNase-A treatment at 37°C for 1 hr and proteinase K treatments for 3 hrs at 60°C. The recovered DNA was further purified with a QIAquick PCR purification kit (Qiagen), with elution in 15µl of Qiagen Elution Buffer (alternatively, Phenol-chloroform extraction and ethanol precipitation).
Briefly for library construction, ChIP DNA fragments were end-repaired and ligated to indexed sequencing adapters (NEB or Rubicon library kit) and amplified with 8-11 rounds of PCR, depending on the DNA amount.  Using SPRI beads (Beckmancoulter), small fragments (<200bp) were removed from the amplified library. The size-selected library was sequenced to 50bp single-end read lengths on an Illumina HiSeq4000 (Vincent J. Coates Genomic Sequencing Laboratory, UC Berkeley).

value: Brain

value: 6 weeks


title: Standard TKS390A G4 Thymus WT input (replica 2)

extract_protocol:
For standard ChIP-seq, the protocol published by Kitagawa et al. (Nat Immunol 2017, 18, 173-183) was basically followed with minor modifications.  Thymocytes prepared from thymus of Satb1+/+  mice (P13)were cross-linked with 1% formaldehyde for 10 min at room temperature. After quenching cross-linking reaction (0.125M Gly at final), these cells were lysed in buffer (4% SDS, 50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 100 mM NaCl) with Protease inhibitor (Roche) and RNase inhibitor. Cross-linked DNA was sonicated using Branson sonifier (20% power, 10 sec on and 30 sec off for 4-6 cycles) in sonication buffer (0.5% Na-laurylsarcosine, 0.1% DOC, 1mM EDTA, 0.5mM EGTA, 10mM Tris-HCl, 100mM NaCl) with Protease inhibitor and RNase inhibitor to generate fragment size 300-1,000bp. DNAs was reverse-crosslinked at 65oC overnight, followed by RNase-A treatment and proteinase K treatments. The recovered DNA was further purified with a QIAquick PCR purification kit (Qiagen), with elution in 15µl of Qiagen Elution Buffer (alternatively, Phenol-chloroform extraction and ethanol precipitation). The purified DNAs were subjected to sequencing library construction.
Briefly for library construction, DNA fragments were end-repaired and ligated to indexed sequencing adapters (NEB or Rubicon library kit) and amplified with 8-11 rounds of PCR, depending on the DNA amount.  Using SPRI beads (Beckmancoulter), small fragments (<200bp) were removed from the amplified library. The size-selected library was sequenced to 50bp single-end read lengths on an Illumina HiSeq4000 (Vincent J. Coates Genomic Sequencing Laboratory, UC Berkeley).

value: Thymus


title: LNA#1:D7 ES Control ChIP-seq input, rep1

status: Public on Dec 01 2021

city: Boston

first: Hyun Jung

last: Oh

state: MA

address: 185 Cambridge St.

institute: MGH

data_processing:
ChIP-Seq: ChIP-seq reads were subjected to the trimming of adapter sequence and removal of duplicates using Trim Galore! (v0.4.1) and cutadapt (v1.2.1) and then mapped to CAST/EiJ (Mus m. castaneus; cas) and 129S1/SvImJ (Mus m. musculus; mus) genomes using Novoalign (v3.00.02) with the parameters (-i300 100 -t180 -F STDFQ -rRandom -h180 180 -v180). From the independent mapping to cas and mus reads, we generated composite (sum of neutral, cas-specific and mus-specific) reads mapped back to the mm9 genome using a previously published pipeline (Pinter et al. Spreading of X chromosome inactivation via a hierarchy of defined Polycomb stations. 2012). Composite bam files having only uniquely aligned reads were used. Log2 fold-enrichment estimates (relative to input) coverage (bigWig) files for ChIP were generated using SPP software with a window of 150 bp and step size 25 bp (get.conservative.fold.enrichment.profile). Coverage (bigWig) of input was normalized by FPM (Fragments Per Million mapped reads) scaling factor.
Genome_build: mm9
Supplementary_files_format_and_content: Log2 fold-enrichment estimates (relative to input) coverage (bigWig) files generated by SPP. FPM-normalized coverage (bigWig) files for input.

source_name: Day 7 ES cells

growth_protocol: Undifferentiated female mouse embryonic stem cells were grown on the irradiated MEF feeders in regular DMEM medium (high-glucose) with 15% FBS and LIF. For differentiation, cells were trypsinizied and incubated in the medium at 37 °C for at least 40 min. After separation of ES cells from attached feeders, ES cells were re-plated on non-gelatinized petri dishes in the complete DMEM medium without LIF. Embryonic bodies (EB) formed from ES cells were transferred to the gelatinized cell culture plates on day 4 of differentiation. Cells were harvested on indicated days.

extract_protocol: Nuclei chromatin was prepared from the cells crosslinked with 1% formaldehyde and sheared by sonication. CTCF ChIP was performed using CTCF antibody (Cell Signaling, #2899S). Library construction was performed using  NEBNext® ChIP-Seq Library Prep Master Mix Set for Illumina.

treatment_protocol: LNA transfection: ES cells on day 7 of differentiation were trypsinized, transfected with scrambled LNA or Jpx LNA#1, transferred to the gelatinized plates and harvested after incubation at 37°C  for 8 hr.

value: CAST/EiJ x 129S1

value: Differentiating mESC (day 7)

tag: transfection

value: Scrambled LNA-transfected

tag: molecule subtype

value: Input DNA


title: D7 ES Jpx-AS CHART-seq, rep1

data_processing:
Library strategy: CHART-seq
CHART-Seq: CHART-seq reads were subjected to the trimming of adapter sequence and removal of duplicates using Trim Galore! (v0.4.1) and cutadapt (v1.2.1) and then mapped to CAST/EiJ (Mus m. castaneus; cas) and 129S1/SvImJ (Mus m. musculus; mus) genomes using Novoalign (v3.00.02) with the parameters (-i300 100 -t180 -F STDFQ -rRandom -h180 180 -v180). From the independent mapping to cas and mus reads, we generated composite (sum of neutral, cas-specific and mus-specific) reads mapped back to the mm9 genome using a previously published pipeline (Pinter et al. Spreading of X chromosome inactivation via a hierarchy of defined Polycomb stations. 2012). Composite bam files having only uniquely aligned reads were used. The input-subtracted coverage (bigWig) files for each CHART experiment were generated using SPP software with a window of 500 bp and step size 100 bp (get.smoothed.tag.density). Coverage (bigWig) of input was normalized by FPM (Fragments Per Million mapped reads) scaling factor.
Genome_build: mm9
Supplementary_files_format_and_content: Input-subtracted coverage (bigWig) files generated by SPP. FPM-normalized coverage (bigWig) files are provided for input.

extract_protocol: CHART-seq: Cells were crosslinked with 1% formaldehyde and nuclei were further crosslinked with 3% formaldehyde. Nuclei chromatin was sheared by sonication to yield the majority of fragments with approximately 3kb. The resulting extracts were hybridized with either Jpx CHART probes (biotinylated DNA oligonucleotides that were complementary to Jpx) or Jpx AS CHART probes (the reverse complement of Jpx CHART probes) and then captured by streptavidin beads. After washes, DNA was eluted in the elution buffer with RNase H. No-RNaseH CHART was generated from Jpx CHART eluant in the absence of RNase H. CHART enriched DNA was further sonicated and subjected to library construction steps including end-repair, dA-tailing, adaptor ligation, double-size selection and PCR amplification using NEBNext® ChIP-Seq Library Prep Master Mix Set for Illumina.

tag: cell type

value: Jpx AS CHART-enriched DNA


title: LNA#1:D7 ES Jpx KD ChIP-seq CTCF, rep2

treatment_protocol: LNA transfection: ES cells on day 7 of differentiation were trypsinized, transfected with scrambled LNA or Jpx LNA#1 by nucleofection, transferred to the gelatinized plates and harvested after incubation at 37°C  for 8 hr.

tag: strain

value: Jpx LNA#1-transfected

value: CTCF ChIP-enriched DNA


title: D7 ES CHART-seq input, rep1


title: LNA#1:D7 ES Jpx KD ChIP-seq CTCF, rep1


title: LNA#1:D7 ES Control ChIP-seq CTCF, rep2


title: LNA#1:D7 ES Jpx KD RNA-seq, rep1

data_processing:
RNA-seq: RNA-seq reads were adapter-trimmed using Trim Galore! (v0.4.1) and cutadapt (v1.2.1), and then aligned to the mm9 genome using TopHat2 (v2.0.10) (--mate-inner-dist 100 --mate-std-dev 100 --library-type fr-firststrand). Only uniquely mapped reads were selected and FPM (Fragments Per Million mapped reads)-normalized coverage (bigWig) files were generated for forward or reverse strand.
Genome_build: mm9
Supplementary_files_format_and_content: FPM-normalized coverage (bigWig) files for forward or reverse strand.

extract_protocol: Total RNA was extracted using TRIzol, depleted of ribosomal RNA and then treated with DNase. The rRNA-depleted RNA was selected by size greater than 200nt, fragmented to a mediam size of about 200nt and then subjected to RT reaction. The subsequent first-strand cDNA was subjected to second strand cDNA synthesis using NEBNext Ultra Directional RNA Second Strand Synthesis Module (NEB, #E7550S).  Double-stranded DNA was subjected to library construction using  NEBNext® ChIP-Seq Library Prep Master Mix Set for Illumina.

value: rRNA-depleted total RNA


title: LNA#1:D7 ES Jpx KD RNA-seq, rep2


title: LNA#1:D7 ES Jpx KD ChIP-seq input, rep1


title: D7 ES Jpx CHART-seq, rep1

value: Jpx CHART-enriched DNA


title: D3 ES CHART-seq input, rep1

source_name: Day 3 ES cells

value: Differentiating mESC (day 3)


title: LNA#1:D7 ES Control RNA-seq, rep2


title: LNA#1:D7 ES Control RNA-seq, rep1


title: Urea TKS355B P13 Thymus wt Satb1 1583D 2ul (replica 2)

extract_protocol:
Thymocytes isolated from thymus of Satb1+/+ mice (P13)  were cross-linked with 1% formaldehyde for 10 min at room temperature, followed by addition of glycine at 0.125 M for 5 min to quench cross-linking reaction. These cells were washed with 1X PBS, suspended in 1% BSA-PBS and cells were precipitated in a tube.  These cells were re-suspended in lysis buffer (4% SDS, 50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 100 mM NaCl) with Protease inhibitor (Roche) and RNase inhibitor.  The resulting cell lysate was incubated for 30 min at room temperature and loaded on top of 8 M urea followed by ultracentrifugation in a Beckman TL-100 Ultracentrifuge for 4-5 hrs at 45K rpm (181,000g, g=relative centrifugation force or RCF; TLS55 rotor).  Recovered chromatin pellet was sonicated to generate fragment size ranging 300–1,000bp using Branson sonifier (20% power, 10 sec on and 30 sec off for 4-6 cycles) in 300ml of sonication buffer [0.5% Na-laurylsarcosine, 0.1% deoxycholate (DOC), 1mM EDTA, 0.5mM EGTA, 10mM TrisHCl, 100mM NaCl] with Protease inhibitor and RNase inhibitor. Note: urea-purified crosslinked chromatin is highly vulnerable to sonication, therefore, it needs to adjust the sonication condition depending on cell type and/or the size of the precipitate DNA.  The sonicated chromatin with 1% Triton X-100 were dialyzed against dialysis buffer (10 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 5% glycerol), and dialyzed for 16 hrs at 4°C (this dialysis is optional).  The chromatin sample was centrifuged at 15,000g for 5 min at 4°C to remove a trace amount of residual insoluble components.   The test-reverse cross-linking of chromatin aliquot (5-10ml of the final volume) was done at 65°C for 2-3 hrs in buffer containing 1% SDS, 100 mM NaCl, 1 mM EDTA, 10mMTris.  Samples were further treated by 100 µg/ml RNase A at 37°C for 1 hr, 200 µg/ml proteinase K at 60°C for more than 3 hrs, followed by DNA purification using QIAquick PCR purification kit (Qiagen), and shearing efficiency was examined by Bioanalyzer 2100 (Agilent Technologies Inc.). For chromatin-immunoprecipitation, 1 µg of an antibody of choice were added to 10 ~ 20 µg of sonicated solubilized chromatin in 350 ml in dilution buffer (1% Triton X-100, 0.01% SDS, 1 mM EDTA, 15 mM Tris-HCl, 100 mM NaCl) and incubated overnight with rotation at 4°C.  At least three chromatin samples were prepared for ChIP-seq from independent experiments to confirm the results. The chromatin fragments bound to antibodies (after incubation overnight) were captured using 20 µl of protein-A, protein-G or protein AG Dynabeads (Invitrogen; pre-washed with 0.1% BSA [Fraction V, Sigma), by 4 hrs incubation time at 4°C.  Dynabeads bound to chromatin fragments were washed twice with Binding buffer (10 mM Tris, pH8.0, 1 mM EDTA, 1 mM EGTA, 0.5% Triton X-100, 0.1% DOC, 100 mM NaCl), twice with LiCl buffer (100 mM Tris [pH 8.0], 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, and 0.5% DOC).  The tubes were changed and washed twice with 0.1M NaCl-TE buffer. The immunoprecipitated samples were eluted from Dynabeads by elution buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 100 mM NaCl and 1% SDS) at 65oC for 20 min.  After elution, ChIP DNAs was reverse-crosslinked at 65oC for 6 hrs, followed by RNase-A treatment at 37°C for 1 hr and proteinase K treatments for 3 hrs at 60°C. The recovered DNA was further purified with a QIAquick PCR purification kit (Qiagen), with elution in 15µl of Qiagen Elution Buffer (alternatively, Phenol-chloroform extraction and ethanol precipitation).
Briefly for library construction, ChIP DNA fragments were end-repaired and ligated to indexed sequencing adapters (NEB or Rubicon library kit) and amplified with 8-11 rounds of PCR, depending on the DNA amount.  Using SPRI beads (Beckmancoulter), small fragments (<200bp) were removed from the amplified library. The size-selected library was sequenced to 50bp single-end read lengths on an Illumina HiSeq4000 (Vincent J. Coates Genomic Sequencing Laboratory, UC Berkeley).

value: Satb1 1583D

value: home made

tag: genotype

value: SATB1 Wild-type

value: urea purified chromatin