title: WT_THAL-SNS-032_C

data_processing:
conversion and demultiplexing: Illumina2bam
FASTQ conversion: bamtools v2.3.0 convert -format fastq
trimming: BBMAP v38.00 'bbduk.sh -ref "polyA.fa.gz","truseq.fa.gz" k=13 ktrim=r useshortkmers=t mink=5 qtrim=r trimq=10 minlength=20'
alignment: STAR v2.5.2b to a concatenated hg38_SIRV-Set-3 index with '--outFilterType BySJout --outFilterMultimapNmax 20 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 999 --outFilterMismatchNoverLmax 0.6 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --outSAMattributes NH HI NM MD --outSAMtype BAM SortedByCoordinate'
read counting: 'htseq-count v0.6.0 -m intersection-nonempty -s yes -f bam -r pos' on a hg38_SIRV-Set-3.gtf annotation
normalization: LOESS normalization was performed based on SIRV-Set-3 spike-ins using R library affy v1.50.0 normalize.loess
Genome_build: hg38
Supplementary_files_format_and_content: bigWig generation: strand-specific signal tracks were computed with 'bedtools v2.26.0 genomeCoverageBed -bg -strand' followed by UCSC 'bedGraphToBigWig v4'

source_name: KBM7_wild-type_6h 250nM THAL-SNS-032 (dCDK9)

growth_protocol: KBM7 cells were grown in IMDM medium supplemented with 10% FBS and 1% Pen/Strep. 48h prior to treatment, all cell lines were seeded to 500k cells/mL in 10mL to T25 flasks. For treatment, 800k cells/condition were seeded in 1mL to 24-well plates in triplicates.

extract_protocol:
Total RNA was isolated using commercially available kits (RNeasy mini, Qiagen). For cell-count normalization, equal amounts (10µL of 1:100 dilution = 0.303ng) of synthetic mRNA spike-ins (SIRV-Set-3; Lexogen, Vienna, Austria) were added to QIAshredder-homogenized lysates after the first extraction step with buffer RLT. Total RNA was quantified using NanoDrop and diluted to 70ng/uL for library preparation.
Quant-seq libraries were generated from 350ng RNA using a commercially available kit (Lexogen, Vienna, Austria). Samples were PCR-amplified for 13 cycles, yielding 0.67-3.16ng/µL DNA per sample. Samples were pooled to equimolar amounts for 50bp sequencing in single-read mode.

treatment_protocol: Cells were treated for 6h with 500nM dTAG7, 100nM dBET6, 250nM THAL-SNS-032 (dCDK9) from 1000x stocks or DMSO. Cells were quantitatively harvested by centrifugation, washed with PBS and snap-frozen in liquid nitrogen.

value: wild-type

value: 6h 250nM THAL-SNS-032 (dCDK9)


title: JH4_CTCFdegron_NT_a_rep6

status: Public on May 11 2020

type: SRA

city: Boston

first: Frederick

middle: W

last: Alt

state: MA

address: 1 Blackfan Circle

department: PCMM

country: USA

institute: Boston Children's Hospital

description: aligned to AJ851868/mm9 hybrid genome

library_source: genomic

data_processing:
Library strategy: Linear-amplification PCR-mediated HTGTS
Standard basecalling formats for Miseq reads
Miseq reads were de-multiplexed and adapter sequence trimmed using the fastq-multx tool from ea-utils (http://code.google.com/p/eautils/) and the SeqPrep utility (https://github.com/jstjohn/SeqPrep) respectively.
Reads were mapped to the indicated reference genome using Bowtie2 (http://bowtiebio.sourceforge.net/bowtie2/manual.shtml) with the top fifty alignments reported that had an alignment score above 50, representing a perfect 25nt local alignment.
We used a best-path searching algorithm to select the optimal sequence of alignments that describe the read’s composition. Aligned reads were filtered on the following conditions: (1) reads must include both a bait alignment and a prey alignment and (2) the bait alignment cannot extend more than 10 nucleotides beyond the targeted site. For vector controls and offset nicking with multiple sites, the longest targeted site was used.
We compared discarded alignments to the selected prey alignment; if any of the discarded alignments surpassed both a coverage and score threshold with respect to the prey alignment, the read was filtered due to low mapping quality.
To remove possible mispriming events and other artifacts, the bait alignment must extend 10 nucleotides past the primer.
Post-filter stringency was applied to remove background-prone junctions with gaps larger than 30nt, bait sequences shorter than 50nt and sequences with microhomology larger than 5 bp.
Genome_build: mm9, mm9_DFL16.1-RSS-U-inversion, mm9_DFL16.1-RSS-D-inversion, mm9_DFL16.1-inversion, mm9_DQ52-inversion, mm9_DH-cluster-inversion, mm9_DQ52-replace-DFL16.1,  mm9_DQ52inv-replace-DFL16.1, mm9_DFL16.1-replace-DQ52, mm9_DFL16.1inv-replace-DQ52, mm9_DFL16.1JH4
Supplementary_files_format_and_content: tab delimited text files contain filtered unique junctions and include the following information: sequence ID (Qname); prey chromosome (Rname), prey junction coordinate (Junction), chromosome orientation of prey junction (Strand), beginning (Rstart) and end (Rend) nucleotide position of prey junction aligning to the genome build; bait chromosome (B_Rname); beginning (B_Rstart) and end (B_Rend) nucleotide position of the bait junction; chromosome orientation of the bait junction (B_Strand); position on the read where the bait sequence begins (B_Qstart) and ends (B_Qend); position on the read where the prey junction begins (Qstart) and ends (Qend); the length of the combined and stitched paried end read (Qlen); the entire stitched paired end read sequence (Seq); the sequence in the + orientation at the junction (J_seq); and the experiment associated with the read (Library)

molecule: genomic DNA

organism: Mus musculus

source_name: v- Abl transformed pro-B cell line

extract_protocol:
DNA was lysed overnight at 56degC in a lysis buffer containing: 200mM NaCl, 0.4% SDS, 100mM Tris-HCl pH7.5, 5mM EDTA pH8.0, 200ug/mL proteinase K, precipitated by isopropanol and dissolved in buffer containing 10mM Tris-HCl pH7.5, 0.1mM EDTA
LAM-HTGTS V(D)J-Seq libraries were prepared using linear-amplification PCR-mediated HTGTS.

treatment_protocol: 3 uM STI-571 treated for 4 days

tag: tissue

value: v- Abl transformed pro-B cell line

tag: genotype

value: CTCFdegron, Emu-Bcl2, RAG2 deficient

tag: treatment protocol

value: RAG2 nucleofected, 3 uM STI-571 treated for 4 days

tag: growth protocol


title: WT_THAL-SNS-032_B

city: Wien

first: Georg

middle: E

last: Winter

state: Austria

address: Lazarettgasse 14, AKH BT25.3

country: Austria

institute: CeMM - Research Institute for Molecular Medicine of the Austrian Academy of Sciences

library_source: transcriptomic

molecule: polyA RNA

organism: Homo sapiens

tag: cell line

tag: cell type

value: chronic myeloid leukemia cell line

tag: treatment


title: 3C_iEm_Rad21degron_IAA_rep2

description: aligned to AJ851868/mm9 hybrid genome

data_processing:
Standard basecalling formats for Miseq reads
Miseq reads were de-multiplexed and adapter sequence trimmed using the fastq-multx tool from ea-utils (http://code.google.com/p/eautils/) and the SeqPrep utility (https://github.com/jstjohn/SeqPrep) respectively.
Reads were mapped to inditated reference genome using Bowtie2 (http://bowtiebio.sourceforge.net/bowtie2/manual.shtml) with the top fifty alignments reported that had an alignment score above 50, representing a perfect 25nt local alignment.
We used a best-path searching algorithm to select the optimal sequence of alignments that describe the read’s composition. Aligned reads were filtered on the following conditions: (1) reads must include both a bait alignment and a prey alignment and (2) the bait alignment cannot extend more than 10 nucleotides beyond the targeted site. For vector controls and offset nicking with multiple sites, the longest targeted site was used.
We compared discarded alignments to the selected prey alignment; if any of the discarded alignments surpassed both a coverage and score threshold with respect to the prey alignment, the read was filtered due to low mapping quality.
To remove possible mispriming events and other artifacts, the bait alignment must extend 10 nucleotides past the primer.
Post-filter stringency was applied to remove background-prone junctions with gaps larger than 30nt, bait sequences shorter than 50nt and sequences with microhomology larger than 5 bp.
Genome_build: AJ851868/mm9 hybrid genome
Supplementary_files_format_and_content: bedgraph files

source_name: v-Abl transformed pro-B cell line

extract_protocol:
DNA was lysed overnight at 56degC in a lysis buffer containing: 200mM NaCl, 0.4% SDS, 100mM Tris-HCl pH7.5, 5mM EDTA pH8.0, 200ug/mL proteinase K, precipitated by isopropanol and dissolved in buffer containing 10mM Tris-HCl pH7.5, 0.1mM EDTA
3C-HTGTS libraries were prepared using linear-amplification PCR-mediated HTGTS.
Linear-amplificate PCR-mediated HTGTS

value: Rad21degron, Emu-Bcl2, RAG2 deficient

value: 500 uM IAA 6h, 3 uM STI-571 with 500uM IAA treated for 4 days


title: 3C_iEm_Rad21degron_NT_rep2

description: aligned to AJ851868/mm9 hybrid genome

value: 3 uM STI-571 treated for 4 days


title: PRO-seq_MJ-19-30_MED14-dTAG_2h_dTAG7_A

description: cross-compare only to MJ-19-30 samples

data_processing:
library strategy: PROT-seq
conversion and demultiplexing: Illumina2bam
FASTQ conversion: bamtools v2.3.0 convert -format fastq
trimming: cutadapt v1.9.1 -a "TGGAATTCTCGGGTGCCAAGG" --minimum-length=10
reverse_complement: fastx_reverse_complement v0.0.14
alignment: 'bowtie v2.2.9 --very-sensitive' to a fused index of hg38_dm6_rDNA-U13369; later filtered with 'samtools v1.7 view -q 20'
spike-in_counting: number of reads aligning to dm6 were counted with "samtools view | awk '$3 ~ /dm6_/ {++dm6_count}'" and normalization factor alpha=1e6/dm6_count (see PMID: 25437568)
signal_trimming_to_3prime_base: only 3' base (Pol II active center) was retained in BED files with "bedtools v2.26.0 bamtobed | awk '($6 == "+") {print $1,$3-1,$3,$4,$5,$6}; ($6 == "-") {print $1,$2,$2+1,$4,$5,$6}'"
Genome_build: hg38
Supplementary_files_format_and_content: bigWig generation: spike-in normalized, strand-specific signal tracks were computed with 'bedtools v2.26.0 genomeCoverageBed -bg -strand -scale $alpha' followed by UCSC 'bedGraphToBigWig v4'

molecule: total RNA

growth_protocol: KBM7 cells were grown in IMDM medium supplemented with 10% FBS and 1% Pen/Strep.

extract_protocol:
PRO-seq libraries were generated as previously described (PMID: 27442863), with 4 parallel run-on reactions per sample. Total RNA was merged after the first RNA precipitation step.
Library construction by PCR amplification with custom primers.

treatment_protocol: 60mio cells were treated for 0.5-2h with 500nM dTAG7, 500nM NVP2, or DMSO and 5% Drosophila S2 cells added for cross-normalization before extracting nuclei on ice.

tag: genotype/variation

value: 2h 500nM dTAG7

tag: experiment id

tag: molecule subtype

value: nuclear RNA


title: del_chd1_ATAC-seq_2

city: Daejeon

first: Hyelim

last: Jo

address: 335 Gwahangno, Yuseong-Gu

department: Biological science

country: South Korea

institute: KAIST

data_processing:
Alignment: bowtie2 (Index; Saccer3)
Sorting and converting to bam files: samtools
Visualization: HOMER
Converting to BigWig files: bedGraphToBigWig
Genome_build: Saccer3
Supplementary_files_format_and_content: BigWig files of the processed ChIP-seq&ATAC-seq raw files and Wig files of the MNase-seq

organism: Saccharomyces cerevisiae

growth_protocol: Yeast strains were grown at 30℃ in  YP medium containing 2% glucose.

extract_protocol:
The amplified library was purified with a Qiagen PCR purification kit.
ChIP-seq libraries for ChIP-seq were prepared 10ng of input DNA or 1 to  10 ng of eluted ChIP DNA using NEXT-flex ChIP-seq kit(Bioo Scientific,5143). ATAC-seq libraries were prepared customized adapter primer. The libraries for MNase-seq were prepared using ase-seq were prepared alsing and TruSeq Nano DNA LT Library Prer Kit(Illumina, FC-121-4001/4002). 100ng of Mononucleosome

treatment_protocol: spheroplasted with 50 mg/ml zymolyase 20T, and then incubated with transposase. Transposition reactions and PCR amplifications were performed.

tag: background genotype

tag: antibody


title: JH4_CTCFdegron_NT_b_rep5

description: aligned to AJ851868/mm9 hybrid genome


title: 3C_VH81X_Rad21degron_IAA_rep2

description: aligned to AJ851868/mm9 hybrid genome


title: WT_THAL-SNS-032_A


title: JH4_BMProB_rep5

description: aligned to AJ851868/mm9 hybrid genome

source_name: 129SV mouse BM pro-B cell

value: 129SV mouse BM pro-B cell

value: Wild-type

value: Sorted pro-B cells by FACS (B220+CD43+IgM-)


title: JH4_CTCFdegron_NT_a_rep5

description: aligned to AJ851868/mm9 hybrid genome


title: del_chd1_ATAC-seq_1


title: WT_DMSO_C

source_name: KBM7_wild-type_6h DMSO

value: 6h DMSO


title: 3C_VH81X_Rad21degron_NT_rep2

description: aligned to AJ851868/mm9 hybrid genome


title: JH4_BMProB_rep4

description: aligned to AJ851868/mm9 hybrid genome


title: WT_ATAC-seq_1

value: wild type(w303a)


title: JH4_CTCFdegron_NT_a_rep3

description: aligned to AJ851868/mm9 hybrid genome


title: WT_dTAG_7_A

source_name: KBM7_wild-type_6h 500nM dTAG7

value: 6h 500nM dTAG7


title: WT_dTAG_7_C

value: wild-type


title: JH4_CTCFdegron_NT_b_rep3

description: aligned to AJ851868/mm9 hybrid genome


title: CTCF_degron_IAA_GRO_rep1

description: aligned to AJ851868/mm9 hybrid genome

data_processing:
Library strategy: Gro-Seq
Reads were aligned using bowtie 2.2.8 with --non-deterministic flag
reads were quantified for specific loci using samtools version 1.8
bigwig files were produced using RseqQ package
Genome_build: AJ851868/mm9 hybrid genome
Supplementary_files_format_and_content: bigwig files contain peak information for samples

extract_protocol:
Briefly, 10 million cells were permeabilized with permeabilization buffer (10 mM Tris-HCl, pH 7.4, 300 mM sucrose, 10 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.05% Tween-20, 0.1% NP40 substitute, 0.5 mM DTT, one tablet of protease inhibitors per 50 ml and 4 units Rnase inhibitor per ml) and resuspended in 100 ul storage buffer (10 mM Tris-HCl, pHHH 8.0, 25% (vol/vol) glycerol, 5 mM MgCl2, 0.1 mM EDTA and 5 mM DTT). Permeabilized cells were subjected to nuclear run-on (2xRun-on mix: 5 mM Tris-Cl, PH 8.0, 2.5 mM MgCl2, 0.5 mM DTT, 150 mM KCl, 0.5 mM ATP, 0.5 mM CTP, 0.5 mM GTP, 0.5 mM BrUTP, 10 units of RNase inhibitor, 1% sarkosyl) at 37o for 5 min, followed by Trizol-based RNA extraction.
Illumina adapters were ligated to RNA followed by cDNA generation with reverse transcriptase.  Libraries were PCR-amplified with Illumina primers and subjected to sequencing.


title: WT_ATAC-seq_2


title: CTCFChIP_CTCFdegron_IAA_input_rep1

description: aligned to AJ851868/mm9 hybrid genome

data_processing:
Reads were aligned using bowtie 2.2.8 with --non-deterministic flag
reads were quantified for specific loci using samtools version 1.8
bigwig files were produced using RseqQ package
Genome_build: AJ851868/mm9 hybrid genome,hg19
Supplementary_files_format_and_content: bigwig files contain peak information for samples

extract_protocol:
Crosslinked cells were treated with cell lysis buffer (5mM PIPES pH 8, 85mM KCl, 0.5% NP-40) and nuclei lysis buffer (50mM TrisCl pH 8.1, 10mM EDTA, 1% SDS) and subjected to sonication. IP and Input DNA were de-crosslinked at 65o overnight and purified via Qiagen PCR purification columns.
ChIP-Seq libraries were prepared using NEBNext Ultra II DNA Library Prep Kit (NEB, #E7645)


title: JH4_CTCFdegron_NT_a_rep4

description: aligned to AJ851868/mm9 hybrid genome


title: JH4_CTCFdegron_NT_b_rep2

description: aligned to AJ851868/mm9 hybrid genome


title: WT_dTAG_7_B


title: JH4_CTCFdegron_NT_b_rep4

description: aligned to AJ851868/mm9 hybrid genome


title: WT_DMSO_B


title: CTCFChIP_parental_input_rep1

description: aligned to AJ851868/mm9 hybrid genome

value: Emu-Bcl2, RAG2 deficient


title: PRO-seq_MJ-19-14_MED14-dTAG_1h_DMSO_A

description: cross-compare only to MJ-19-14 samples

value: 1h DMSO


title: WT_sua7_input_ChIP-seq_2


title: WT_DMSO_A


title: JH4_CTCFdegron_NT_a_rep2

description: aligned to AJ851868/mm9 hybrid genome

value: v- Abl transformed pro-B cell line

value: CTCFdegron, Emu-Bcl2, RAG2 deficient


title: Rad21ChIP_CTCFdegron_IAA_rep1

description: aligned to AJ851868/mm9 hybrid genome

value: Rad21 antibody (Abcam, #ab992)


title: del_chd1_sua7_input_ChIP-seq_2


title: Parental_GRO_rep1

description: aligned to AJ851868/mm9 hybrid genome


title: del_chd1_sua7_input_ChIP-seq_1


title: WT_dBET6_C

source_name: KBM7_wild-type_6h 100nM dBET6

value: chronic myeloid leukemia cell line

value: 6h 100nM dBET6


title: JH4_CTCFdegron_NT_b_rep1

description: aligned to AJ851868/mm9 hybrid genome

value: RAG2 nucleofected, 3 uM STI-571 treated for 4 days


title: CTCFChIP_CTCFdegron_IAA_rep1

description: aligned to AJ851868/mm9 hybrid genome

value: 500 uM IAA 6h, 3 uM STI-571 with 500uM IAA treated for 4 days

value: CTCF antibody (Millipore, #07-729)


title: WT_dBET6_B


title: JH4_CTCFdegron_NT_a_rep1

description: aligned to AJ851868/mm9 hybrid genome


title: CTCFChIP_parental_NT_rep1

description: aligned to AJ851868/mm9 hybrid genome

value: 3 uM STI-571 treated for 4 days


title: JH4_CTCFtagging_only_IAA_b_rep4

description: aligned to AJ851868/mm9 hybrid genome

value: CTCF-mAID-GFP tagging, Emu-Bcl2, RAG2 deficient

value: RAG2 nucleofected,  500 uM IAA 6h, 3 uM STI-571 with 500uM IAA treated for 4 days


title: JH4_CTCFtagging_only_IAA_a_rep4

description: aligned to AJ851868/mm9 hybrid genome


title: del_sua7-Flag_input_ChIP-seq_1

value: FLAG(Sigma)


title: MED6_dTAG_7_C

source_name: KBM7_MED6-dTAG_6h 500nM dTAG7


title: WT_dBET6_A


title: Rad21ChIP_HCT116spikingin_Rad21degron_IAA_input_rep1

description: aligned to AJ851868/mm9 hybrid genome

value: Rad21degron, Emu-Bcl2, RAG2 deficient


title: MED6_dTAG_7_B

value: 6h 500nM dTAG7


title: WT_sua7-Flag_ChIP-seq_2


title: Rad21ChIP_HCT116spikingin_Rad21degron_NT_input_rep1

description: aligned to AJ851868/mm9 hybrid genome


title: Rad21ChIP_HCT116spikingin_Rad21degron_IAA_rep1

description: aligned to AJ851868/mm9 hybrid genome


title: JH4_CTCFtagging_only_IAA_a_rep3

description: aligned to AJ851868/mm9 hybrid genome


title: notag_for_Flag_ChIP-seq_2

value: wild type(w303a)


title: WT_sua7-Flag_ChIP-seq_1


title: MED6_DMSO_C

source_name: KBM7_MED6-dTAG_6h DMSO

value: 6h DMSO


title: WT_sua7_input_ChIP-seq_1


title: MED6_dTAG_7_A


title: JH4_parental_IAA_rep1_offtarget

description: aligned to AJ851868/mm9 hybrid genome

value: WT, Emu-Bcl2, RAG2 deficient


title: notag_for_Flag_ChIP-seq_1


title: del_chd1_input_ChIP-seq_2


title: Rad21ChIP_parental_rep1

description: aligned to AJ851868/mm9 hybrid genome

value: Emu-Bcl2, RAG2 deficient


title: JH4_CTCFtagging_only_IAA_b_rep2

description: aligned to AJ851868/mm9 hybrid genome

value: v- Abl transformed pro-B cell line


title: del_chd1_sua7-Flag_ChIP-seq_2


title: Rad21ChIP_HCT116spikingin_Rad21degron_NT_rep1

description: aligned to AJ851868/mm9 hybrid genome

value: Rad21 antibody (Abcam, #ab992)


title: MED6_DMSO_B

value: chronic myeloid leukemia cell line


title: JH4_CTCFtagging_only_IAA_a_rep2

description: aligned to AJ851868/mm9 hybrid genome


title: 3C_iEm_CTCFdegron_IAA_rep1

description: aligned to AJ851868/mm9 hybrid genome

value: CTCFdegron, Emu-Bcl2, RAG2 deficient


title: MED6_DMSO_A


title: JH4_CTCFtagging_only_IAA_b_rep3

description: aligned to AJ851868/mm9 hybrid genome


title: del_chd1_input_ChIP-seq_1


title: MED31_dTAG_7_C

source_name: KBM7_MED31-dTAG_6h 500nM dTAG7


title: JH4_CTCFtagging_only_IAA_a_rep1

description: aligned to AJ851868/mm9 hybrid genome


title: JH4_CTCFtagging_only_IAA_b_rep1

description: aligned to AJ851868/mm9 hybrid genome


title: WT_input_ChIP-seq_2


title: MED31_dTAG_7_B


title: 3C_VH81X_Rad21degron_NT_rep1

description: aligned to AJ851868/mm9 hybrid genome


title: JH4_CTCFtagging_only_NT_b_rep4

description: aligned to AJ851868/mm9 hybrid genome

value: RAG2 nucleofected, 3 uM STI-571 treated for 4 days


title: JH4_CTCFtagging_only_NT_b_rep3

description: aligned to AJ851868/mm9 hybrid genome


title: MED31_DMSO_C

source_name: KBM7_MED31-dTAG_6h DMSO


title: MED31_DMSO_B


title: MED31_dTAG_7_A


title: WT_Pser2_ChIP-seq_2

value: RNAPII CTD Ser2p


title: 3C_VH81X_Rad21degron_IAA_rep1

description: aligned to AJ851868/mm9 hybrid genome

value: 500 uM IAA 6h, 3 uM STI-571 with 500uM IAA treated for 4 days


title: WT_Pser2_ChIP-seq_1


title: JH4_CTCFtagging_only_NT_a_rep4

description: aligned to AJ851868/mm9 hybrid genome


title: del_chd1_Pser2_ChIP-seq_1


title: 1049473.Intrahepatic.HT29.Liver.Tumor.SCID.B37 and mm10

city: Tarrytown

first: Alexandra

last: Eichten

state: NY

address: 777 old saw mill river road

institute: Regeneron Pharmaceuticals Inc.

description: male

data_processing:
The Cell Ranger Suite version 2.0 was used to perform sample de-multiplexing, barcode processing and single cell gene UMI (unique molecular index)
Genome_build: mm10 for mouse cells and B37 for human cells
Supplementary_files_format_and_content: tab-delimited text files include UMI values for samples/cells

organism:
Homo sapiens
Mus musculus

source_name: Intrahepatic HT-29 Liver+Tumor

growth_protocol: HDD with 50 μg of plasmids encoding mKras (G12D), Cas9 and sgTrp53 or mKras (G12D), Cas9 and sgRNA (empty vector) was used to induce liver tumors in CB.17/SCID, C57Bl6 and Balb/c mice; 1x10^6 HT-29 human colon carcinoma cells were grown intrahepatically in male CB.17/SCID mice.

extract_protocol:
Tissues (tumor bearing liver (tumor and adjacent normal), adjacent normal, liver tumors and naïve liver) were minced, enzymatically digested for 13 minutes using 2.5 mg/ml collagenase Type II, 2.5 mg/ml collagenase Type IV (Gibco), and 0.5 mg/ml DNase (Sigma) in PBS / 1% BSA at 37°C, treated with DMEM containing 10% FBS and filtered (70-µm nylon filter; Falcon). Single cell suspensions were subjected to 1X PharMLyse for 5 (tumor) or 10 (tumor bearing liver, adjacent normal tissue, naïve liver tissue) minutes, washed and subjected to dead cell removal kit (130-090-101, Miltenyi). The remaining cells were used for single cell sequencing.
Single cell suspensions from HT-29 tumor bearing livers underwent a depletion step using the following FITC conjugated Abs: single cell suspensions were incubated with anti-CD44 (338804, Biolegend) and anti-EpCam (324204, Biolegend) antibodies using 10 µl anti-FITC-beads for 1x10^6 total cells. The remaining cells were used for single cell sequencing.
Cellular suspensions (~6000 cells) were loaded on a Chromium Single Cell Instrument (10X Genomics) to generate single cell GEMs. Single-cell RNAseq libraries were prepared using version 1 Chromium Single cell 3’ Library, Gel beads & Mutiplex kit (10X Genomics).

treatment_protocol: no treatments

tag: strain

value: SCID

value: Intrahepatic HT-29 Liver+Tumor

tag: age

value: adult

value: WT


title: JH4_CTCFtagging_only_NT_a_rep3

description: aligned to AJ851868/mm9 hybrid genome

value: CTCF-mAID-GFP tagging, Emu-Bcl2, RAG2 deficient


title: del_chd1_Pser2_ChIP-seq_2


title: 3C_iEm_Rad21degron_IAA_rep1

description: aligned to AJ851868/mm9 hybrid genome

value: Rad21degron, Emu-Bcl2, RAG2 deficient


title: 3C_iEm_parental_rep1

description: aligned to AJ851868/mm9 hybrid genome

value: 3 uM STI-571 treated for 4 days


title: 1049470.Intrahepatic.HT29.Tumor.SCID.B37 and mm10

description: male

source_name: Intrahepatic HT-29 Tumor

value: Intrahepatic HT-29 Tumor


title: 3C_iEm_Rad21degron_NT_rep1

description: aligned to AJ851868/mm9 hybrid genome


title: WT_H3K36Ac_ChIP-seq_2

value: wild type(w303a)


title: JH4_CTCFtagging_only_NT_b_rep2

description: aligned to AJ851868/mm9 hybrid genome


title: WT_H3K36Ac_ChIP-seq_1


title: JH4_CTCFtagging_only_NT_a_rep2

description: aligned to AJ851868/mm9 hybrid genome


title: WT_input_ChIP-seq_1


title: 1049468.Intrahepatic.HT29.Liver.Tumor.SCID.B37 and mm10

description: male


title: MED31_DMSO_A

value: 6h DMSO


title: MED28_dTAG_7_B

source_name: KBM7_MED28-dTAG_6h 500nM dTAG7

value: 6h 500nM dTAG7


title: MED28_dTAG_7_A


title: 1049467.Intrahepatic.HT29.Tumor.SCID.B37 and mm10

description: male


title: WT_H3_ChIP-seq_1


title: JH4_CTCFtagging_only_NT_a_rep1

description: aligned to AJ851868/mm9 hybrid genome

value: v- Abl transformed pro-B cell line


title: MED28_DMSO_C

source_name: KBM7_MED28-dTAG_6h DMSO

value: chronic myeloid leukemia cell line


title: 1049466.Intrahepatic.HT29.Liver.SCID.B37 and mm10

description: male

source_name: Intrahepatic HT-29 Liver

value: Intrahepatic HT-29 Liver


title: YFP+IgD- Ezh2(Y641F)fl/wt;Cγ1-cre 3

city: New York

first: Cem

last: Meydan

state: New York

address: 1305 York

department: Physiology & Biophysics

institute: Weill Cornell Medical College

description: cells sorted: YFP+,IgD-

data_processing:
Cell ranger 3.0.2 was used to process the fastq files per 10X specifications.
Genome_build: mm10
Supplementary_files_format_and_content: Matrix of gene counts per cell in hdf5 format

source_name: germinal center and post-germinal center B-cells from spleen

extract_protocol:
Cells were sorted into PBS and ten thousand cells from each spleen were subjected to single cell RNA-seq using 10X Genomics Chromium platform
Library preparation for single cell 3’ RNA-seq v2, sequencing and post-processing of the raw data was performed at the Epigenomics Core at Weill Cornell Medicine. Libraries were prepared according to 10X Genomics specifications.

treatment_protocol: Mice were immunized with sheep red blood cells (SRBC) 8 days before extraction of spleens

value: 10 weeks

tag: littermate

value: litter 1


title: YFP+IgD- Ezh2(Y641F)fl/wt;Cγ1-cre 2

description: cells sorted: YFP+,IgD-


title: JH4_Ostir_only_IAA_b_rep4

description: aligned to AJ851868/mm9 hybrid genome

value: Ostir1 knock in,Emu-Bcl2, RAG2 deficient

value: RAG2 nucleofected,  500 uM IAA 6h, 3 uM STI-571 with 500uM IAA treated for 4 days


title: MED28_DMSO_A


title: JH4_Ostir_only_IAA_b_rep3

description: aligned to AJ851868/mm9 hybrid genome


title: JH4_Ostir_only_IAA_a_rep4

description: aligned to AJ851868/mm9 hybrid genome


title: MED28_DMSO_B


title: MED28_dTAG_7_C


title: MED26_dTAG_7_C

source_name: KBM7_MED26-dTAG_6h 500nM dTAG7


title: WT_H3_ChIP-seq_2


title: YFP+IgD- Ezh2(Y641F)fl/wt;Cγ1-cre 1

description: cells sorted: YFP+,IgD-


title: 1049469.Intrahepatic.HT29.Liver.SCID.B37 and mm10

description: male

value: SCID


title: JH4_Ostir_only_IAA_a_rep3

description: aligned to AJ851868/mm9 hybrid genome

source_name: v- Abl transformed pro-B cell line


title: YFP+IgD- Cγ1-cre 3

description: cells sorted: YFP+,IgD-


title: JH4_Ostir_only_IAA_b_rep2

description: aligned to AJ851868/mm9 hybrid genome


title: MED26_dTAG_7_B


title: 244-2-4

type: RNA

city: Lund

first: Jan

last: Marsal

address: BMC D14

country: Sweden

institute: Lund University

hyb_protocol: Processing of samples for microarray hybridization was carried out at KFB as described in the Affymetrix GeneChip Whole Transcript (WT) Sense Target Labeling Assay manual.

data_processing: Samples were subjected to the Affymetrix Human Gene 2.1 ST Array and raw data was analyzed using R (version 3.3.0) with Bioconductor pachage xps. Probe level expression values were normalized and summated using Robust Multiarray Analysis (RMA) algorithm based on the gene model for probesets supported by RefSeq and full-length GenBank transcripts, and with a background correction using Epanechnikov Kernel and only the perfect match probes. Finally, expression data were summarized on the level of human gene symbols and finally normalized by quantile normalization with the help of R-package limma.

label: biotin

extract_protocol: Gut biopsies were collected in RNAlater Stabilization Solution (Ambion, Applied Biosystems) and then frozen on dry ice. Samples were sent on dry ice to KFB Center of Excellence for Fluorescent Bioanalytics (Regensburg, Germany) for extracting materials.

tag: status

value: Uninflamed-after


title: YFP+IgD- Cγ1-cre 2

description: cells sorted: YFP+,IgD-


title: JH4_CTCFtagging_only_NT_b_rep1

description: aligned to AJ851868/mm9 hybrid genome

value: RAG2 nucleofected, 3 uM STI-571 treated for 4 days


title: JH4_Ostir_only_IAA_a_rep2

description: aligned to AJ851868/mm9 hybrid genome


title: MED26_DMSO_C

source_name: KBM7_MED26-dTAG_6h DMSO


title: YFP+IgD- Cγ1-cre 1

description: cells sorted: YFP+,IgD-


title: JH4_Ostir_only_IAA_b_rep1

description: aligned to AJ851868/mm9 hybrid genome


title: 244-2-3

scan_protocol: Processing of samples for microarray hybridization was carried out at KFB as described in the Affymetrix GeneChip Whole Transcript (WT) Sense Target Labeling Assay manual.

value: Inflamed-after


title: JH4_Ostir_only_IAA_a_rep1

description: aligned to AJ851868/mm9 hybrid genome


title: T9S4

city: Fukuoka

first: Yasuyuki

last: Ohkawa

address: 3-1-1 Maidashi

country: Japan

institute: Medical Institute of Bioregulation

data_processing:
The raw sequence reads were aligned to the rat reference genome (Rnor_6.0) using HISAT2 (version 2.0.4).
Read counting was performed with HTSeq (version 0.6.0) so that UMI duplicates were removed.
Genome_build: Rnor_6.0
Supplementary_files_format_and_content: The txt file contains UMI count matrix.

organism: Rattus norvegicus

source_name: Spinal cord

extract_protocol:
Spinal cords were removed, flash frozen in liquid nitrogen, and RNA was harvested using NucleoSpin® RNA XS (TaKaRa).
Libraries were constructed by using CEL-Seq2 protocol.
RNA-Seq (3' tag-Seq)

tag: group

value: 5 days-post injury with epidural electrical spinal stimulation

value: T9 spinal cord

value: Sprague Dawley


title: 244-2-2

source_name: Ileum

label_protocol: Processing of samples for microarray hybridization was carried out at KFB as described in the Affymetrix GeneChip Whole Transcript (WT) Sense Target Labeling Assay manual.


title: MED26_DMSO_B


title: MED26_dTAG_7_A


title: MED14_dTAG_7_C

source_name: KBM7_MED14-dTAG_6h 500nM dTAG7


title: T9S2

tag: tissue


title: JH4_Ostir_only_NT_b_rep4

description: aligned to AJ851868/mm9 hybrid genome

tag: genotype


title: T9S3


title: MED26_DMSO_A


title: JH4_Ostir_only_NT_a_rep4

description: aligned to AJ851868/mm9 hybrid genome

tag: treatment protocol


title: JH4_Ostir_only_NT_b_rep3

description: aligned to AJ851868/mm9 hybrid genome

tag: growth protocol


title: MED14_dTAG_7_B

tag: cell line


title: MED14_dTAG_7_A

tag: cell type


title: T9I4

value: 5 days-post injury


title: T9I3


title: JH4_Ostir_only_NT_a_rep3

description: aligned to AJ851868/mm9 hybrid genome


title: MED14_DMSO_C

source_name: KBM7_MED14-dTAG_6h DMSO

tag: treatment

value: 6h DMSO


title: 244-2-1


title: JH4_Ostir_only_NT_a_rep2

description: aligned to AJ851868/mm9 hybrid genome


title: MED14_DMSO_A


title: MED14_DMSO_B


title: 244-1-3

value: Inflamed-before


title: JH4_Ostir_only_NT_b_rep2

description: aligned to AJ851868/mm9 hybrid genome

value: v- Abl transformed pro-B cell line


title: 244-1-4

value: Uninflamed-before


title: 244-2-5


title: MED12_dTAG_7_C

source_name: KBM7_MED12-dTAG_6h 500nM dTAG7

value: chronic myeloid leukemia cell line

value: 6h 500nM dTAG7


title: T9S1


title: T9I1


title: 241-2-4


title: 244-1-1


title: T9U4

value: without spinal cord injury


title: JH4_Ostir_only_NT_a_rep1

description: aligned to AJ851868/mm9 hybrid genome

value: Ostir1 knock in,Emu-Bcl2, RAG2 deficient


title: JH4_Ostir_only_NT_b_rep1

description: aligned to AJ851868/mm9 hybrid genome


title: JH4_parental_IAA_b_rep4

description: aligned to AJ851868/mm9 hybrid genome

value: WT, Emu-Bcl2, RAG2 deficient

value: RAG2 nucleofected,  500 uM IAA 6h, 3 uM STI-571 with 500uM IAA treated for 4 days


title: T9U2


title: MED12_dTAG_7_A


title: 241-2-2

value: Uninflamed-after


title: 241-1-3


title: 241-1-2


title: 241-1-4


title: 244-1-5


title: MED12_DMSO_B

source_name: KBM7_MED12-dTAG_6h DMSO


title: T9U1


title: MED12_DMSO_A


title: JH4_parental_IAA_b_rep3

description: aligned to AJ851868/mm9 hybrid genome


title: T9U3


title: MED12_DMSO_C


title: 241-2-1


title: JH4_parental_IAA_a_rep3

description: aligned to AJ851868/mm9 hybrid genome


title: JH4_parental_IAA_b_rep2

description: aligned to AJ851868/mm9 hybrid genome


title: DASH_3500_35000_Takara_Salmonella

city: Würzburg

first: Tom

last: Gräfenhan

address: Josef-Schneider-Str. 2, Bau D15

department: Core Unit SysMed

country: Germany

institute: University of Wuerzburg

data_processing:
FastQ reads trimmed for the adapter sequences using Cutadapt version 2.5 with the –nextseq-trim=20
Mapping with segemehl via READemption with the argument –a 80 (NEB samples) or –a 80 –R (Takara samples)
Gene quantification via READemption subcommand gene_quanti with arguments –a –o 10
wig files for coverage plots were generated via READemption subcommand coverage
Genome_build: Salmonella (NC_016810.1, NC_017718.1, NC_017719.1, NC_017720.1); Bacteroides (NC_004663.1, NC_004703.1)
Supplementary_files_format_and_content: Tab-delimited text files include read count values for each sample
Supplementary_files_format_and_content: wig files

organism: Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344

source_name: whole cells

growth_protocol: Salmonella was grown until OD 2.0; Bacteroides until OD 0.5.

extract_protocol:
Cultures were pelleted by centrifugation and RNA was extracted with Trizol. To remove contaminating genomic DNA, samples were further treated with 0.25 U of DNase I (Fermentas) per 1 µg of RNA for 45 min at 37°C, followed by phenol-chloroform extraction and ethanol precipitation of RNA.
Libraries were generated with NEBNext Magnesium RNA Fragmentation Module (# E6150S) in combination with NEB NEBNext Multiplex Small RNA Library Prep Kit for Illumina (#E7560S) or Takara SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (#634411). Where indicated, ribosomal cDNA was depleted by the DASH procedure: Depletion of Abundant Sequences by Hybridization using target specific sgRNAs and Cas9 to remove unwanted high-abundance species in sequencing libraries.

tag: growth phase

value: Exponential

tag: agent

value: DASH

tag: library prep kit

value: Takara SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian #634411

tag: rna template in rt (ng)

tag: sgrna pool size

tag: pre-incubation

value: yes

tag: cdna

tag: cdna removal

value: Proteinase K


title: T9I2

value: 5 days-post injury

value: T9 spinal cord

tag: strain

value: Sprague Dawley


title: 241-1-1

tag: status


title: MED10_dTAG_7_C

source_name: KBM7_MED10-dTAG_6h 500nM dTAG7


title: noDASH_Takara_Salmonella


title: DASH_3500_35000_NEB_Salmonella

value: NEB NEBNext  Multiplex Small RNA Library Prep Kit for Illumina  #E7560S


title: 241-2-3


title: 244-1-2

value: Inflamed-before


title: MED12_dTAG_7_B

value: chronic myeloid leukemia cell line


title: 240-2-2

source_name: Caecum

value: Inflamed-after


title: MED10_dTAG_7_B

value: 6h 500nM dTAG7


title: 240-2-3


title: JH4_parental_IAA_a_rep2

description: aligned to AJ851868/mm9 hybrid genome

tag: tissue

value: v- Abl transformed pro-B cell line


title: DASH_100_1000_NEB_Salmonella


title: 240-2-1


title: JH4_parental_IAA_b_rep1

description: aligned to AJ851868/mm9 hybrid genome

tag: genotype

value: WT, Emu-Bcl2, RAG2 deficient


title: JH4_parental_IAA_a_rep1

description: aligned to AJ851868/mm9 hybrid genome

tag: treatment protocol

value: RAG2 nucleofected,  500 uM IAA 6h, 3 uM STI-571 with 500uM IAA treated for 4 days


title: MED10_DMSO_B

source_name: KBM7_MED10-dTAG_6h DMSO

value: 6h DMSO


title: DASH_1000_10000_NEB_Salmonella

value: Exponential


title: MED10_DMSO_C


title: noDASH_1_PK


title: DASH_1_PK

value: DASH


title: JH4_parental_NT_a_rep4

description: aligned to AJ851868/mm9 hybrid genome

value: RAG2 nucleofected, 3 uM STI-571 treated for 4 days

tag: growth protocol


title: MED1_dTAG_7_C

source_name: KBM7_MED1-dTAG_6h 500nM dTAG7

tag: cell line


title: noDASH_NEB_Salmonella


title: MED10_dTAG_7_A

tag: cell type


title: MED10_DMSO_A

tag: treatment


title: JH4_parental_NT_b_rep4

description: aligned to AJ851868/mm9 hybrid genome


title: JH4_parental_NT_a_rep3

description: aligned to AJ851868/mm9 hybrid genome


title: 240-1-3

value: Uninflamed-before


title: 240-1-1


title: noDASH_10_PK

tag: growth phase

value: Proteinase K


title: JH4_CTCFdegron_IAA_b_rep6

description: aligned to AJ851868/mm9 hybrid genome

data_processing:
Standard basecalling formats for Miseq reads
Miseq reads were de-multiplexed and adapter sequence trimmed using the fastq-multx tool from ea-utils (http://code.google.com/p/eautils/) and the SeqPrep utility (https://github.com/jstjohn/SeqPrep) respectively.
Reads were mapped to the indicated reference genome using Bowtie2 (http://bowtiebio.sourceforge.net/bowtie2/manual.shtml) with the top fifty alignments reported that had an alignment score above 50, representing a perfect 25nt local alignment.
We used a best-path searching algorithm to select the optimal sequence of alignments that describe the read’s composition. Aligned reads were filtered on the following conditions: (1) reads must include both a bait alignment and a prey alignment and (2) the bait alignment cannot extend more than 10 nucleotides beyond the targeted site. For vector controls and offset nicking with multiple sites, the longest targeted site was used.
We compared discarded alignments to the selected prey alignment; if any of the discarded alignments surpassed both a coverage and score threshold with respect to the prey alignment, the read was filtered due to low mapping quality.
To remove possible mispriming events and other artifacts, the bait alignment must extend 10 nucleotides past the primer.
Post-filter stringency was applied to remove background-prone junctions with gaps larger than 30nt, bait sequences shorter than 50nt and sequences with microhomology larger than 5 bp.
Genome_build: AJ851868/mm9 hybrid genome
Supplementary_files_format_and_content: tab delimited text files contain filtered unique junctions and include the following information: sequence ID (Qname); prey chromosome (Rname), prey junction coordinate (Junction), chromosome orientation of prey junction (Strand), beginning (Rstart) and end (Rend) nucleotide position of prey junction aligning to the genome build; bait chromosome (B_Rname); beginning (B_Rstart) and end (B_Rend) nucleotide position of the bait junction; chromosome orientation of the bait junction (B_Strand); position on the read where the bait sequence begins (B_Qstart) and ends (B_Qend); position on the read where the prey junction begins (Qstart) and ends (Qend); the length of the combined and stitched paried end read (Qlen); the entire stitched paired end read sequence (Seq); the sequence in the + orientation at the junction (J_seq); and the experiment associated with the read (Library)

source_name: v-Abl transformed pro-B cell line

extract_protocol:
DNA was lysed overnight at 56degC in a lysis buffer containing: 200mM NaCl, 0.4% SDS, 100mM Tris-HCl pH7.5, 5mM EDTA pH8.0, 200ug/mL proteinase K, precipitated by isopropanol and dissolved in buffer containing 10mM Tris-HCl pH7.5, 0.1mM EDTA
LAM-HTGTS V(D)J-Seq libraries were prepared using linear-amplification PCR-mediated HTGTS.
Linear-amplification PCR-mediated HTGTS

value: v-Abl transformed pro-B cell line

value: CTCFdegron, Emu-Bcl2, RAG2 deficient


title: 240-1-4


title: 239-2-2

value: Uninflamed-after


title: JH4_CTCFdegron_IAA_a_rep6

description: aligned to AJ851868/mm9 hybrid genome


title: 240-1-2


title: MED1_dTAG_7_A


title: noDASH_100_PK

tag: agent

value: NEB NEBNext  Multiplex Small RNA Library Prep Kit for Illumina  #E7560S


title: 239-2-1


title: MED1_DMSO_B

source_name: KBM7_MED1-dTAG_6h DMSO


title: noDASH_1000_PK

tag: library prep kit


title: JH4_CTCFdegron_IAA_b_rep5

description: aligned to AJ851868/mm9 hybrid genome


title: JH4_CTCFdegron_IAA_a_rep5

description: aligned to AJ851868/mm9 hybrid genome


title: CDK9_dTAG_7_C

source_name: KBM7_CDK9-dTAG_6h 500nM dTAG7


title: JH4_CTCFdegron_IAA_b_rep4

description: aligned to AJ851868/mm9 hybrid genome


title: DASH_10_PK

tag: rna template in rt (ng)

value: yes


title: DASH_100_PK

tag: sgrna pool size


title: DASH_1000_PK

tag: pre-incubation


title: noDASH_1_C

tag: cdna

value: Column purification


title: MED1_dTAG_7_B

value: chronic myeloid leukemia cell line


title: 239-1-1-repeat

value: Inflamed-before


title: 237-2-4


title: CDK9_dTAG_7_B

value: 6h 500nM dTAG7


title: 239-1-2


title: DASH_1_C

tag: cdna removal


title: 237-2-3


title: JH4_Rad21degron_deIGCR1_IAA_b_rep4

description: aligned to AJ851868/mm9 hybrid genome

value: IGCR1deleted, Rad21degron, Emu-Bcl2, RAG2 deficient


title: MED1_DMSO_A


title: MED1_DMSO_C


title: JH4_parental_IAA_a_rep4

description: aligned to AJ851868/mm9 hybrid genome

value: v- Abl transformed pro-B cell line

value: WT, Emu-Bcl2, RAG2 deficient

value: RAG2 nucleofected,  500 uM IAA 6h, 3 uM STI-571 with 500uM IAA treated for 4 days


title: noDASH_10_C

tag: strain

value: Exponential


title: CDK9_dTAG_7_A


title: JH4_Rad21degron_deIGCR1_NT_b_rep4

description: aligned to AJ851868/mm9 hybrid genome

value: IGCR1deleted,Rad21degron, Emu-Bcl2, RAG2 deficient

value: RAG2 nucleofected, 3 uM STI-571 treated for 4 days


title: CDK9_DMSO_C

source_name: KBM7_CDK9-dTAG_6h DMSO

value: 6h DMSO


title: 237-2-1

tag: status


title: 239-1-1


title: CDK9_DMSO_B


title: JH4_CTCFdegron_IAA_a_rep4

description: aligned to AJ851868/mm9 hybrid genome

tag: tissue

value: v-Abl transformed pro-B cell line


title: JH4_CTCFdegron_IAA_b_rep3

description: aligned to AJ851868/mm9 hybrid genome

tag: genotype

value: CTCFdegron, Emu-Bcl2, RAG2 deficient


title: noDASH_100_C


title: DASH_10_C

value: DASH


title: JH4_parental_NT_b_rep3

description: aligned to AJ851868/mm9 hybrid genome

source_name: v- Abl transformed pro-B cell line

tag: treatment protocol


title: 237-1-3

value: Uninflamed-before


title: JH4_CTCFdegron_IAA_a_rep3

description: aligned to AJ851868/mm9 hybrid genome


title: CDK9_DMSO_A


title: noDASH_1000_C

value: NEB NEBNext  Multiplex Small RNA Library Prep Kit for Illumina  #E7560S


title: LDK6_BulkSeq_ColdDiss_Fresh

city: Perth

first: Alistair

middle: R. R.

last: Forrest

state: WA

address: 6 Verdun Street, Nedlands

country: Australia

institute: Harry Perkins Institute of Medical Research, the University of Western Australia

data_processing:
Bulk RNA-seq data processing  BCL files were demultiplexed and converted into FASTQ using bcl2fastq utility of Illumina BaseSpace Sequence Hub. FastQC was used for read quality control (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Adapters and low-quality bases were trimmed using Trim Galore with parameters --paired --quality 5 --stringency 5 --length 20 --max_n 10 (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). Reads matching to ribosomal DNA repeat sequence BK000964 (https://www.ncbi.nlm.nih.gov/nuccore/BK000964) and low complexity reads were removed with TagDust2 (Lassmann, 2015). The remaining reads were mapped to GRCm38.84 version of mouse genome using STAR version 2.6.1a with default settings (Dobin et al., 2013). Picard MarkDuplicates tool was employed to identify duplicates (https://broadinstitute.github.io/picard/command-line-overview.html#MarkDuplicates). FeatureCounts was then used to derive gene count matrix (Liao, Smyth, & Shi, 2014). Counts were normalised to gene length and then to library sizes using weighted trimmed mean of M-values (TMM) method in edgeR (Robinson, McCarthy, & Smyth, 2010), to derive gene length corrected trimmed mean of M-values (GeTMM) as described in (Smid et al., 2018).  scRNA-seq and snRNA-seq data processing  BCL files were demultiplexed and converted into FASTQ using bcl2fastq utility of Illumina BaseSpace Sequence Hub. scRNA-seq and snRNA-seq libraries were processed using Cell Ranger 2.1.1 with mm10-2.1.0 reference. Reads mapped to exons were used for scRNA-seq samples, whereas both intronic and exonic reads were counted for snRNA-seq. Custom pre-mRNA reference for snRNA-seq was built as described in https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/advanced/references#premrna. Raw gene-barcode matrices from Cell Ranger output were used for downstream processing. Cells were distinguished from background noise using EmptyDrops (Lun et al., 2019). Only genes detected in a minimum of 10 cells were retained; cells with 200-3000 genes and under 50% of mitochondrial reads were retained, as per Park et al. study (Park et al., 2018). Nuclei were additionally filtered to have at least 450 UMIs for v2 chemistry and 900 UMIs for v3 chemistry, and mitochondrial genes were removed. Outlier cells with high ratio of number of detected UMI to genes (>3 median absolute deviations from median) were removed using Scater (McCarthy, Campbell, Lun, & Wills, 2017). Seurat v2 was used for sample integration (canonical correlation analysis), normalisation (dividing by the total counts, multiplying by 10,000 and natural-log transforming), scaling, clustering and differential expression analysis (Wilcoxon test) (Butler, Hoffman, Smibert, Papalexi, & Satija, 2018).
Genome_build: mm10
Supplementary_files_format_and_content: Tab-delimited text files include raw gene counts for each individual library. For bulk RNA-seq libraries, files include one column with counts for the corresponding sample. For single-cell and single-nuclei libraries, files include multiple columns where each column corresponds to one cell or nuclei.

source_name: adult whole kidney

extract_protocol:
Cold tissue dissociation  Kidneys were dissociated using a modified version of the published protocol described in (Adam, Potter, & Potter, 2017). Based on the weight, in a pre-cooled Miltenyi C-tube, a protease solution (5mM CaCl2 [Invitrogen; AM9530G], 10mg/mL B. Licheniformis protease [Sigma; P5380], 125U/mL DNase I [Sigma; D5025], 1xDPBS) was prepared for each kidney.  The kidneys were then minced on ice into a smooth paste using a scalpel. The minced kidney was transferred into 4-6mL of the protease solution (dependent on weight) and triturated using a 1mL pipette for 15 seconds every two minutes for a total of eight minutes.  Following trituration, the C-tubes were placed onto a Miltenyi gentleMACS octo dissociator in a cool room (4°C), and the m_brain_03 program was run twice in succession. Once complete, the samples were triturated for 15 seconds every two minutes on ice for an additional 16 minutes using a 1mL pipette. 10µL of each sample was then loaded into a haemocytometer to assess whether tissue dissociation was complete. The dissociated cells were transferred to a 15mL centrifuge tube and 3mL of ice-cold PBS+10%FBS [Gibco; A3160401] was added.  The cell suspension was centrifuged at 1200g for five minutes at 4°C. The supernatant was removed and the pellet was resuspended in 2mL of PBS+10%FBS. The cells were then filtered through a 70µm cell strainer, which was subsequently rinsed with 2mL of PBS+0.01% BSA. The cells were then centrifuged again at 1200g for five minutes at 4°C followed by removal of the supernatant and resuspension of the pellet in 5mL of PBS+0.01%BSA. The cells were then filtered through a 40µm cell strainer, which was subsequently rinsed with 2mL of PBS+0.01% BSA. The cells were again centrifuged at 1200g for five minutes at 4°C followed by removal of the supernatant and resuspension of the cells in 5mL of PBS+0.04%BSA. The cells were counted and checked for viability using the ReadyProbes Blue/Red Kit on the Countess II FL. Bulk RNA-seq was performed using the NEBNext Ultra II RNA Library Kit for Illumina [NEB; E7760] and NEBNext Poly(A) mRNA isolation module [NEB; E7490] as described in the manufacturers’ protocol, with 100ng of total RNA as input.
Refer to extract protocol for each individual file
Single cell libraries prepared using 10x 3’ v2&v3 kits following the manufacturers protocol. Bulk libraries were prepared using the NEBNext ultra II directional RNA library prep kit for Illumina following the manufacturers protocol. For the bulk libraries, rRNA was removed using either poly(A)+ selection using the NEBNext Poly(A) mRNA magnetic isolation module [E7490], or the NEBNext rRNA depletion kit (human/mouse/rat) kit [E6310].

value: Balb/c

tag: Sex

value: Female

tag: workflow

value: Bulk RNA-seq of cold-dissociated fresh cell suspension

tag: sequencing protocol

value: bulk RNA-seq


title: 237-1-4


title: 237-2-2

value: Uninflamed-after


title: JH4_Rad21degron_deIGCR1_NT_b_rep3

description: aligned to AJ851868/mm9 hybrid genome

tag: treatment


title: DASH_1000_C

tag: growth phase

value: yes

value: Column purification


title: LDK4_BulkSeq_ColdDiss_Fresh


title: JH4_BMProB_rep3

description: aligned to AJ851868/mm9 hybrid genome

source_name: 129SV mouse BM pro-B cell

value: 129SV mouse BM pro-B cell

value: Wild-type

value: Sorted pro-B cells by FACS (B220+CD43+IgM-)


title: 237-1-2

source_name: Ileum

value: Inflamed-before


title: LDK5_BulkSeq_ColdDiss_Fresh


title: DASH_preincubation

value: Exponential

tag: agent


title: JH4_Rad21degron_IAA_a_rep3

description: aligned to AJ851868/mm9 hybrid genome

value: v-Abl transformed pro-B cell line

value: Rad21degron, Emu-Bcl2, RAG2 deficient

value: RAG2 nucleofected,  500 uM IAA 6h, 3 uM STI-571 with 500uM IAA treated for 4 days


title: LDK3_Bulk

extract_protocol:
Total RNA was extracted from flash-frozen kidneys using the Nucleospin RNA Midi kit [Macherey Nagel; 740962.20] as per manufacturers’ directions. RNA was assessed for quantity and quality using the TapeStation 4200 RNA ScreenTape kit [Agilent; 5067-5576], which showed all RNA used had a RIN of >8. Bulk RNA-seq was performed using the NEBNext Ultra II RNA Library Kit for Illumina [NEB; E7760] and NEBNext Poly(A) mRNA isolation module [NEB; E7490] as described in the manufacturers’ protocol, with 100ng of total RNA as input.
Refer to extract protocol for each individual file
Single cell libraries prepared using 10x 3’ v2&v3 kits following the manufacturers protocol. Bulk libraries were prepared using the NEBNext ultra II directional RNA library prep kit for Illumina following the manufacturers protocol. For the bulk libraries, rRNA was removed using either poly(A)+ selection using the NEBNext Poly(A) mRNA magnetic isolation module [E7490], or the NEBNext rRNA depletion kit (human/mouse/rat) kit [E6310].

value: Bulk RNA-seq of whole kidney


title: LDK2_Bulk


title: noDASH_preincubation

tag: library prep kit

value: NEB NEBNext  Multiplex Small RNA Library Prep Kit for Illumina  #E7560S


title: LDK1_Bulk

value: Balb/c


title: DASH_100_10

value: DASH

tag: rna template in rt (ng)

value: no


title: JH4_Rad21degron_NT_a_rep3

description: aligned to AJ851868/mm9 hybrid genome

value: RAG2 nucleofected, 3 uM STI-571 treated for 4 days


title: JH4_Rad21degron_NT_b_rep3

description: aligned to AJ851868/mm9 hybrid genome


title: MJ59_SN_sucrose

extract_protocol:
SN_sucrose  Kidneys were removed from -80°C and transferred to a 1.5mL tube containing 500µL of pre-chilled lysis buffer II (same recipe as SN_FANS_3×500g protocols, with 125U/mL of DNase I added) and allowed to rest on ice for two minutes. Each kidney was then homogenised using a pellet pestle with 40 up and down strokes without twisting. The homogenate was transferred to a 15mL centrifuge tube containing 2mL of lysis buffer II and incubated for 12 minutes on ice with mixing every two minutes using a glass fire-polished silanised Pasteur pipette. Following the incubation, 2.5mL of nuclei wash buffer II (1xDPBS+2%BSA) was added to the homogenate. Remaining tissue clumps were dissociated by repeated trituration of the homogenate using the glass Pasteur pipette.  The homogenate was then filtered through a 30µm MACS Smart Strainer into a new 15mL centrifuge tube. Subsequently, the homogenate was centrifuged at 2000g for five minutes at 4°C. The supernatant was removed, and the pellet was resuspended in 510µL of nuclei wash buffer II. 10µL of the suspension was transferred to a 1.5mL tube and placed on ice for use in nuclei recovery calculations. 900µL of 1.8M sucrose solution [Sigma; NUC201] was added to the remaining 500µL of nuclei suspension and homogenised by mixing with a pipette. 3.6mL of 1.3M sucrose solution [Sigma; NUC201] was added to a 5mL tube. The nuclei/sucrose homogenate was then gently layered on top of the 1.3M sucrose solution.   The 5mL tube containing the sucrose solutions and nuclei was then centrifuged at 3000g for 10 minutes at 4°C. Once centrifugation was complete, the sucrose phase containing debris was soaked up using a Kimwipe wrapped around a pellet pestle. The remaining supernatant was removed and discarded using a pipette. The nuclei pellet was then resuspended in 5mL of wash buffer II, of which 10µL was transferred to a 1.5mL tube to assess nuclei recovery.  To the 10µL of nuclei suspension removed prior to the sucrose gradient, 980µL of wash buffer II and 10µL of DAPI (10µg/mL) was added. To the 10µL of nuclei suspension removed after the sucrose gradient, 89µL of wash buffer II and 1µL of DAPI (10µg/mL) was added. The yield from the pre- and post-sucrose aliquots were compared to assess nuclei recovery after filtration through the gradient. The post-sucrose count was used to dilute the nuclei to a concentration of 700nuclei/µL, which was immediately loaded onto a 10x Chip B and processed with the 10x Chromium controller.
Refer to extract protocol for each individual file
Single cell libraries prepared using 10x 3’ v2&v3 kits following the manufacturers protocol. Bulk libraries were prepared using the NEBNext ultra II directional RNA library prep kit for Illumina following the manufacturers protocol. For the bulk libraries, rRNA was removed using either poly(A)+ selection using the NEBNext Poly(A) mRNA magnetic isolation module [E7490], or the NEBNext rRNA depletion kit (human/mouse/rat) kit [E6310].

value: Female

value: Single-nuclei isolation, sucrose gradient

value: snRNA-seq, 10x v3 chemistry


title: DASH_10_100

tag: sgrna pool size

value: Column purification


title: JH4_Rad21degron_IAA_b_rep3

description: aligned to AJ851868/mm9 hybrid genome


title: DASH_1000_100

tag: pre-incubation


title: DASH_1000_1000

tag: cdna


title: 236-2-4

value: Uninflamed-after


title: 236-2-3


title: JH4_BMProB_rep2

description: aligned to AJ851868/mm9 hybrid genome

value: 129SV mouse BM pro-B cell

value: Wild-type

value: Sorted pro-B cells by FACS (B220+CD43+IgM-)


title: 240-2-4

value: Inflamed-after


title: 237-1-1

value: Uninflamed-before


title: 236-1-4


title: DASH_100_100

value: Exponential

tag: cdna removal


title: 236-2-2


title: MJ58_SN_sucrose

tag: strain


title: MJ57_SN_sucrose


title: MJ50_SN_FANS_1×2000g

extract_protocol:
SN_FANS_1x2000g  The flash frozen kidneys were removed from -80°C and transferred to a 1.5mL tube containing 500µL of pre-chilled lysis buffer (same recipe as SN_FANS_3×500g protocol) and allowed to rest on ice for two minutes. Each kidney was then homogenised with a pellet pestle with 40 up and down strokes without twisting the pellet. The resulting homogenate was mixed with a pipette and transferred to pre-cooled 15mL centrifuge tube containing 2mL of lysis buffer. The homogenate was incubated for 12 minutes on ice with mixing every two minutes using a glass fire-polished silanised Pasteur pipette [Kimble; 63A54]. Once incubation was complete, 2.5mL of nuclei wash buffer (same recipe as SN_FANS_3×500g protocol) was added to the homogenate. The remaining tissue fragments were completely dissociated by repeated trituration of the homogenate using the glass Pasteur pipette.  The homogenate was then filtered through a 30µm MACS Smart Strainer [Miltenyi Biotech; 130-098-458] into a new 15mL centrifuge tube. The nuclei were centrifuged at 2000g for five minutes at 4°C. The supernatant was removed and the nuclei pellet was resuspended in 1mL of nuclei wash buffer. 200µL was aliquoted into a 0.5mL tube to be used as an unstained control for sorting. 8µL of DAPI (10µg/mL) was added to the remaining 800µL of nuclei. Quality and quantity of the nuclei was checked using a fluorescence microscope prior to sorting. Sorting and post sorting QC was performed in the same manner as for the SN_FANS_3x500g protocol. Nuclei were then loaded onto a 10x chip B and processed immediately on the 10x Chromium controller.
Refer to extract protocol for each individual file
Single cell libraries prepared using 10x 3’ v2&v3 kits following the manufacturers protocol. Bulk libraries were prepared using the NEBNext ultra II directional RNA library prep kit for Illumina following the manufacturers protocol. For the bulk libraries, rRNA was removed using either poly(A)+ selection using the NEBNext Poly(A) mRNA magnetic isolation module [E7490], or the NEBNext rRNA depletion kit (human/mouse/rat) kit [E6310].

value: Single-nuclei isolation, FANS, one wash and centrifugation speed of 2000g


title: JH4_CTCFdegron_IAA_b_rep2

description: aligned to AJ851868/mm9 hybrid genome

value: v-Abl transformed pro-B cell line

value: CTCFdegron, Emu-Bcl2, RAG2 deficient

value: RAG2 nucleofected,  500 uM IAA 6h, 3 uM STI-571 with 500uM IAA treated for 4 days


title: JH4_parental_NT_b_rep2

description: aligned to AJ851868/mm9 hybrid genome

value: Emu-Bcl2, RAG2 deficient


title: MJ49_SN_FANS_1×2000g

value: Balb/c


title: JH4_parental_NT_a_rep2

description: aligned to AJ851868/mm9 hybrid genome

value: RAG2 nucleofected, 3 uM STI-571 treated for 4 days


title: DASH_100_1000

value: DASH

value: NEB NEBNext  Multiplex Small RNA Library Prep Kit for Illumina  #E7560S

value: no


title: noDASH

value: Column purification


title: MJ48_SN_FANS_1×2000g

value: Female

value: snRNA-seq, 10x v3 chemistry


title: MJ47_SN_FANS_3×500g

extract_protocol:
SN_FANS_3x500g  This method is an adaptation of the Frankenstein protocol (Martelotto, 2019) and the 10x demonstrated protocol (10xGenomics;, 2017).   The kidneys were removed from -80°C and immediately placed on ice. Each kidney was then transferred to a 1.5mL tube containing 300µL of chilled lysis buffer (10mM Tris-HCl [Invitrogen; AM9856], 3mM MgCl2 [Invitrogen; AM9530G], 10mM NaCl [Sigma; 71386], 0.005% Nonidet P40 substitute [Roche; 11754599001], 0.2U/mL RNasin plus) and incubated on ice for two minutes. The tissue was then completely homogenised using a pellet pestle [Fisherbrand; FSB12-141-364] using up and down strokes without twisting. 1.2mL of chilled lysis buffer was added to the tube and pipette-mixed (wide-bore). The full volume was then transferred to a pre-cooled 2mL tube. The homogenate was incubated on ice for five minutes and mixed with a wide-bore tip every 1.5 minutes.  Following the incubation, 500µL of the lysis buffer was added to the homogenate, which was subsequently pipette-mixed and split equally into four 2mL tubes. 1mL of chilled lysis buffer was added to each tube and pipette-mixed using a wide-bore tip. The four tubes were incubated for a further five minutes on ice, mixing with a wide-bore tip every 1.5 minutes. The homogenate from the four tubes was then filtered through a 40µm strainer into a pre-cooled 50ml centrifuge tube. Following this, the sample was split again into four 2mL tubes resting on ice.  The samples were centrifuged at 500g for five minutes at 4°C. The supernatant was removed leaving 50µL in the tube. 1.5mL of lysis buffer was then added to two of the tubes and the pellet resuspended by mixing with a pipette. This resulted in two tubes containing 1.5mL resuspended nuclei in lysis buffer, and two tubes containing a nuclei pellet in 50µL of lysis buffer. The resuspended nuclei in one tube was then combined with the nuclei pellet of another, resulting in two tubes containing resuspended nuclei in lysis buffer.   The nuclei were centrifuged again at 500g for five minutes at 4°C. The supernatant was removed completely and discarded. 500µL of nuclei wash buffer (1xDPBS, 1% BSA, 0.2U/mL RNasin plus) was added to the tube containing the pellet and left to incubate without resuspending for five minutes. Following incubation, an additional 1mL of nuclei wash buffer was added, and the nuclei were resuspended by gently mixing with a pipette. The nuclei were again centrifuged at 500g for five minutes at 4°C, followed by discarding of the supernatant. The pellets were resuspended in 1.4mL of nuclei wash buffer, then transferred into a pre-cooled 1.5mL tube. Another 500g centrifugation step for five minutes at 4°C was performed. The supernatant was then discarded, and the nuclei pellet was resuspended in 1mL of nuclei wash buffer.  The nuclei were then filtered through a 40µm pluristrainer mini filter. 200µL of the filtered nuclei suspension was transferred into a 0.5mL tube and set aside to be used as the unstained control for sorting. To the remaining 800µL, 8µL of DAPI (10µg/mL) [ThermoScientific; 62248] was added, and the nuclei were mixed with a pipette. A quality control step was performed by viewing the nuclei under a fluorescence microscope on a haemocytometer to check nuclei shape and count.   A BD Influx Cell Sorter was then used to sort 100,000 DAPI-positive events using a 70µm nozzle and a pressure of 22 psi (as per gating strategy). The post-sort nuclei concentration and quality were then checked using a fluorescence microscope and haemocytometer. Nuclei were then loaded onto a 10x chip (A/B depending on version used) and processed immediately on the 10x Chromium controller.
Refer to extract protocol for each individual file
Single cell libraries prepared using 10x 3’ v2&v3 kits following the manufacturers protocol. Bulk libraries were prepared using the NEBNext ultra II directional RNA library prep kit for Illumina following the manufacturers protocol. For the bulk libraries, rRNA was removed using either poly(A)+ selection using the NEBNext Poly(A) mRNA magnetic isolation module [E7490], or the NEBNext rRNA depletion kit (human/mouse/rat) kit [E6310].

value: Single-nuclei isolation, FANS, three washes and centrifugation speed of 500g


title: JH4_CTCFdegron_IAA_a_rep2

description: aligned to AJ851868/mm9 hybrid genome


title: JH4_Rad21degron_IAA_b_rep2

description: aligned to AJ851868/mm9 hybrid genome

value: Rad21degron, Emu-Bcl2, RAG2 deficient


title: MJ46_SN_FANS_3×500g


title: MJ45_SN_FANS_3×500g


title: Index_gRNA_libraryI

city: Hangzhou

first: Lijia

last: Ma

state: Zhejiang

address: Shilongshan Road No.18, Cloud Town, Xihu District

department: School of Life Sciences

country: China

institute: Westlake university

data_processing:
The mRNA library(10x Genomics) was processed by CellRanger(v.3.0.1) and aligned to the GRCh38(provided by 10x Genomics) with default alignment parameters.
The gene-barcode matrices was generated by the 'count' command from the mRNA library
Seurat(v.3.1.1) was then applied for further data analysis of clustering and dimensionally reduction. For filtering, cells with more than 7000 or less than 200 detected genes, as well as those with mitochondrial transcripts proportion higher than 35% were excluded
The adapters and low quality bases from Index libraries were removed by using cutadapt(v1.15)
The index libraries were aligned the gRNA library using Bowtie2(v.2.3.5.1)
The number of UMI and reads of each gRNAs for cell were summarized by perl scripts.
Genome_build: GRCh38 and gRNA library
Supplementary_files_format_and_content: mtx, tsv

source_name: Jurkat cells

growth_protocol: Jurkat-Cas9 cells were harvested after 24h of stimulation.Jurkat-Cas9 cells were cultured ten days in  supplemented with 10% heat-inactivated characterized FBS (GEMINI #900-108) , 1% penicillin/streptomycin (Gibco #15140-122), 2ug/ mL puromycin and blasticidine before stimulation.

extract_protocol:
A total of 20×106 Jurkat cells were stimulated by ImmunoCult™ Human CD3/CD28 T Cell Activator (STEMCELL) in RPMI with 10%FBS at the concentration of 5×106/ mL. The activator was added at the dose of 25μl/ mL. After 24h of stimulation, cells were harvested and stained for surface CD69 (FN50, Biolegend), and 2×106 CD69- (Bottom 25%) cell population were sorted using FACS (Fusion, BD) for single cell sequencing.
The mRNA library was prepared following to the Chromium User Guide.

treatment_protocol: A total of 20×106 target cells were infected at MOI≤0.3 in RPMI-1640 containing 8ug/ mL polybrene by centrifuging at 700g, 32oC for 2h. At 48h post transduction, cellular mKate2 expression, indicating the successful transduction, was tested by flow cytometry (Cytoflex, Beckman). Then, the 10 days of puromycin selection started. In the period of puromycin selection, culture medium with puromycin and blasticidine at the concentration of 2ug/ mL was added every two days, and the cell concentration in culture medium was maintained at 5×105/ mL. Meanwhile, cellular mKate2 expression was monitored by flow cytometry until the ratio of mKate2+ cells were higher than 95%, which indicated the end of puromycin selection.

tag: cell type

value: Jurkat-Cas9 cell


title: mRNA_library


title: JH4_Rad21degron_IAA_a_rep2

description: aligned to AJ851868/mm9 hybrid genome

tag: tissue


title: JH4_Rad21degron_deIGCR1_IAA_b_rep2

description: aligned to AJ851868/mm9 hybrid genome

tag: genotype

value: IGCR1deleted, Rad21degron, Emu-Bcl2, RAG2 deficient


title: JH4_Rad21degron_NT_b_rep2

description: aligned to AJ851868/mm9 hybrid genome


title: HBG1_WT

data_processing:
The adapters and low quality bases were removed by using cutadapt (v1.15).
The Fastq files were aligned to the reference genome (hg38) using Hisat2 (v2.1.0).
Reads mapped to tRNA and rRNA are removed.
Stringtie (v2.0.6) was used to calculate the TPM of each gene to represent their mRNA expression level.
The differential genes were identified by a negative binomial model using the DEseq2 package.
Genome_build: hg38
Supplementary_files_format_and_content: txt

source_name: Hemoglobin subunit gamma-1,HBG1, WT-SAM

growth_protocol: The HEK293T-Cas9 cells were harvested on days 3 after transfection and cultured in endotoxin-free DMEM medium with 10% (v/v) fetal bovine serum (FBS, Gibco).

extract_protocol:
Cells were lysed and RNA was extracted using the miRNeasy Mini Kit (Qiagen #217004). cDNA was synthesized using the PrimeScript RT reagent Kit with gDNA Eraser (TAKARA #RR047A) using 200 ng of RNA per cDNA reaction.
RNA libraries were prepared with TruePrep DNA Library Prep Kit V2 for Illumina according to manufacture's instruction

treatment_protocol: Cells were transfected with plasmid.

tag: cell line

value: HEK 293T

tag: rna scaffold target

value: Hemoglobin subunit gamma-1,HBG1 but with WT-SAM


title: JH4_Rad21degron_deIGCR1_NT_b_rep2

description: aligned to AJ851868/mm9 hybrid genome

value: v-Abl transformed pro-B cell line

value: IGCR1deleted,Rad21degron, Emu-Bcl2, RAG2 deficient


title: MJ53_SC_ColdDiss_Cryopreserved

extract_protocol:
Cold tissue dissociation  Kidneys were dissociated using a modified version of the published protocol described in (Adam, Potter, & Potter, 2017). Based on the weight, in a pre-cooled Miltenyi C-tube, a protease solution (5mM CaCl2 [Invitrogen; AM9530G], 10mg/mL B. Licheniformis protease [Sigma; P5380], 125U/mL DNase I [Sigma; D5025], 1xDPBS) was prepared for each kidney.  The kidneys were then minced on ice into a smooth paste using a scalpel. The minced kidney was transferred into 4-6mL of the protease solution (dependent on weight) and triturated using a 1mL pipette for 15 seconds every two minutes for a total of eight minutes.  Following trituration, the C-tubes were placed onto a Miltenyi gentleMACS octo dissociator in a cool room (4°C), and the m_brain_03 program was run twice in succession. Once complete, the samples were triturated for 15 seconds every two minutes on ice for an additional 16 minutes using a 1mL pipette. 10µL of each sample was then loaded into a haemocytometer to assess whether tissue dissociation was complete. The dissociated cells were transferred to a 15mL centrifuge tube and 3mL of ice-cold PBS+10%FBS [Gibco; A3160401] was added.   The cell suspension was centrifuged at 1200g for five minutes at 4°C. The supernatant was removed and the pellet was resuspended in 2mL of PBS+10%FBS. The cells were then filtered through a 70µm cell strainer, which was subsequently rinsed with 2mL of PBS+0.01% BSA. The cells were then centrifuged again at 1200g for five minutes at 4°C followed by removal of the supernatant and resuspension of the pellet in 5mL of PBS+0.01%BSA. The cells were then filtered through a 40µm cell strainer, which was subsequently rinsed with 2mL of PBS+0.01% BSA. The cells were again centrifuged at 1200g for five minutes at 4°C followed by removal of the supernatant and resuspension of the cells in 5mL of PBS+0.04%BSA. The cells were counted and checked for viability using the ReadyProbes Blue/Red Kit on the Countess II FL.  Cryopreservation  Freezing  After tissue dissociation, the cells were centrifuged at 400g for 10 minutes (1200g for five minutes at 4°C for the repeated experiment), then resuspended in freezing media (50% FBS, 40% RPMI-1640 [Gibco; 11875093], 10% DMSO [Sigma; D4540]) to achieve a concentration of 1x106cells/mL. 1mL of the cell suspension was aliquoted into 2mL cryovials, then placed into an isopropanol freezing container (Mr. Frosty) and stored at -80°C overnight. The following day, the cells were transferred to liquid nitrogen storage.  Thawing  The samples were removed from -80°C and immediately placed into a 37°C waterbath for 2-3 minutes to rapidly thaw. The cells were then mixed using a 1mL pipette with a wide-bore tip and the entire volume transferred to a 15mL centrifuge tube [Greiner; 188261]. The cryovial was then rinsed twice with RMPI+10%FBS (rinse media); each time the 1mL of media was added to the 15mL centrifuge in a dropwise manner while gently shaking the tube. 7mL of rinse media was added to the centrifuge tube using a serological pipette – the first 4mL was added dropwise while gently shaking the tube, and the following 3mL added down the side of the tube over two seconds. The tube was then inverted to mix.   The cells were centrifuged at 1200g for five minutes. Once completed, the supernatant was removed (leaving 1mL). 2mL of PBS+0.04% BSA was added to the centrifuge tube and shaken gently to mix. The cells were then centrifuged again at 1200g for five minutes. The supernatant was discarded leaving 0.5mL behind. 0.5mL of PBS+0.04% BSA was added to the cells and gently pipette-mixed 10-15 times to fully resuspend. The cells were then filtered through a pre-wetted (with 1mL of PBS+0.04% BSA) 40µm pluristrainer mini filter. A 20µL aliquot of the cells was used to obtain an estimate of cell count and viability using the ReadyProbes Blue/Red Kit on the Countess II FL. Based on the count, the cells were diluted to a concentration of 700cells/µL. The cells were then loaded onto a 10x chip (A/B depending on version) and immediately processed on the 10x Chromium controller. All single cell libraries were constructed in biological triplicate using the 10x Chromium 3’ workflow as per manufacturers’ directions.
Refer to extract protocol for each individual file
Single cell libraries prepared using 10x 3’ v2&v3 kits following the manufacturers protocol. Bulk libraries were prepared using the NEBNext ultra II directional RNA library prep kit for Illumina following the manufacturers protocol. For the bulk libraries, rRNA was removed using either poly(A)+ selection using the NEBNext Poly(A) mRNA magnetic isolation module [E7490], or the NEBNext rRNA depletion kit (human/mouse/rat) kit [E6310].

tag: Sex

tag: workflow

value: Cold tissue dissociation, cryopreserved suspension

value: scRNA-seq, 10x v3 chemistry


title: DASH_100_C

value: Exponential

value: yes


title: JH4_Rad21degron_NT_a_rep2

description: aligned to AJ851868/mm9 hybrid genome

tag: treatment

value: RAG2 nucleofected, 3 uM STI-571 treated for 4 days


title: MJ52_SC_ColdDiss_Cryopreserved

value: Balb/c

tag: sequencing protocol


title: Index_gRNA_libraryII


title: JH4_Rad21degron_deIGCR1_IAA_b_rep3

description: aligned to AJ851868/mm9 hybrid genome

value: RAG2 nucleofected,  500 uM IAA 6h, 3 uM STI-571 with 500uM IAA treated for 4 days


title: 236-1-3

tag: status

value: Inflamed-before


title: 235-1-4


title: 235-2-4

value: Uninflamed-after


title: 235-1-3


title: 235-2-1


title: 236-2-1


title: 236-1-2


title: HNT_8A8G_Rep2

source_name: non-targeting

value: human non-targeting


title: HBG1_8A8G_Rep1

source_name: Hemoglobin subunit gamma-1,HGB1

value: Hemoglobin subunit gamma-1,HBG1


title: 236-1-1

value: Uninflamed-before


title: 235-2-3


title: 235-1-2-repeat


title: 235-1-2


title: JH4_BMProB_rep1

description: aligned to AJ851868/mm9 hybrid genome

value: 129SV mouse BM pro-B cell

value: Wild-type


title: LD03_SC_ColdDiss_Fresh

extract_protocol:
Cold tissue dissociation  Kidneys were dissociated using a modified version of the published protocol described in (Adam, Potter, & Potter, 2017). Based on the weight, in a pre-cooled Miltenyi C-tube, a protease solution (5mM CaCl2 [Invitrogen; AM9530G], 10mg/mL B. Licheniformis protease [Sigma; P5380], 125U/mL DNase I [Sigma; D5025], 1xDPBS) was prepared for each kidney.  The kidneys were then minced on ice into a smooth paste using a scalpel. The minced kidney was transferred into 4-6mL of the protease solution (dependent on weight) and triturated using a 1mL pipette for 15 seconds every two minutes for a total of eight minutes.  Following trituration, the C-tubes were placed onto a Miltenyi gentleMACS octo dissociator in a cool room (4°C), and the m_brain_03 program was run twice in succession. Once complete, the samples were triturated for 15 seconds every two minutes on ice for an additional 16 minutes using a 1mL pipette. 10µL of each sample was then loaded into a haemocytometer to assess whether tissue dissociation was complete. The dissociated cells were transferred to a 15mL centrifuge tube and 3mL of ice-cold PBS+10%FBS [Gibco; A3160401] was added.   The cell suspension was centrifuged at 1200g for five minutes at 4°C. The supernatant was removed and the pellet was resuspended in 2mL of PBS+10%FBS. The cells were then filtered through a 70µm cell strainer, which was subsequently rinsed with 2mL of PBS+0.01% BSA. The cells were then centrifuged again at 1200g for five minutes at 4°C followed by removal of the supernatant and resuspension of the pellet in 5mL of PBS+0.01%BSA. The cells were then filtered through a 40µm cell strainer, which was subsequently rinsed with 2mL of PBS+0.01% BSA. The cells were again centrifuged at 1200g for five minutes at 4°C followed by removal of the supernatant and resuspension of the cells in 5mL of PBS+0.04%BSA. The cells were counted and checked for viability using the ReadyProbes Blue/Red Kit on the Countess II FL. The cells were further diluted to a concentration of 700cells/µL with PBS/0.04%BSA and loaded directly onto a 10x chip (A/B depending on experiment) and isolated using the 10x Chromium controller. All single cell libraries were constructed in biological triplicate using the 10x Chromium 3’ workflow as per manufacturers’ directions.
Refer to extract protocol for each individual file
Single cell libraries prepared using 10x 3’ v2&v3 kits following the manufacturers protocol. Bulk libraries were prepared using the NEBNext ultra II directional RNA library prep kit for Illumina following the manufacturers protocol. For the bulk libraries, rRNA was removed using either poly(A)+ selection using the NEBNext Poly(A) mRNA magnetic isolation module [E7490], or the NEBNext rRNA depletion kit (human/mouse/rat) kit [E6310].

value: Female

value: Cold tissue dissociation, fresh suspension


title: 3C_iEm_BMProB_rep1

description: aligned to AJ851868/mm9 hybrid genome

growth_protocol: opti-MEM medium with 10% FBS

extract_protocol:
DNA was lysed overnight at 56degC in a lysis buffer containing: 200mM NaCl, 0.4% SDS, 100mM Tris-HCl pH7.5, 5mM EDTA pH8.0, 200ug/mL proteinase K, precipitated by isopropanol and dissolved in buffer containing 10mM Tris-HCl pH7.5, 0.1mM EDTA
3C-HTGTS libraries were prepared using linear-amplification PCR-mediated HTGTS.
Linear-amplificate PCR-mediated HTGTS

value: 2ng/ml IL-7 and 2ng/ml SCF for 4 days


title: MJ51_SC_ColdDiss_Cryopreserved

value: Cold tissue dissociation, cryopreserved suspension


title: 235-2-2

value: Inflamed-after


title: HBG1_8A8G_Rep2

value: HEK 293T


title: LD02_SC_ColdDiss_Fresh

value: scRNA-seq, 10x v3 chemistry


title: 234-2-4-repeat

source_name: Rectum


title: siGLI1_ATAC-seq  rep3

city: Rochester

first: Stephanie

middle: Lynn

last: Safgren

state: Minnesota

address: 200 1st Street SW, Gonda 19 366C

department: Oncology Research

institute: Mayo Clinic

description: Gli1 knockdown

data_processing:
Base calling was performed with the Illumina bcl2fastq
Sequences were trimmed with TrimGalore (v0.4.5), aligned to hg19 (NCBI build 37) using bowtie2 (v2.3.3.1) with maximum fragment length 2000.  Data was sorted to keep only chromosomal reads, remove mitochondrial reads, filtered to remove reads with MAPQ smaller than 30  and duplicates (picard v2.9.0) removed.  Bigwig files were generated with bamCoverage (deepTools 3.3.0) using a bin size of 10, scaling factoer of 0.5 and normalized to RPKM.
Genome_build: hg19

source_name: skeletal muscle

growth_protocol: RMS13 cells grown in RPMI media with 10% fetal bovine supplemented.  Cells were replated before reaching comfluency and media was replaced as needed typically every 2-3 days.

extract_protocol:
DNA for ATAC-seq  was prepared from 20,000 cells following the OMNI-ATAC procedures from Corces et al. (Nature Methods 2017) and Buenrostro et al. (Curr Protoc Mol Biol 2016), with modifications, using the Nextera kit (Illumina).  The cells were lysed for 3 minutes on ice and transposed for 30 minutes at 37C.  DNA was cleaned-up using procedures of a DNA extraction column.
The DNA libraries were prepared with 5-9 cycles of PCR amplification with the NEB High Fidelity Master Mix (New England Biolabs).  Clean up was done using the Zymo DNA Clean and Concentrator kit (Zymo Research) and followed with AMPure XP (Beckman Coulter) bead cleanup to remove primer dimers and under-digested chromatin.

treatment_protocol: siRNA knockdown of Gli1 or SMARCA2 were incubated for 72 hours in standard cell culture conditions.

tag: sirna


title: LD01_SC_ColdDiss_Fresh


title: HNT_8A8G_Rep1


title: siNT2_ATAC-seq  rep3

value: non-targeting


title: MJ13_Bulk

extract_protocol:
Total RNA was extracted from flash-frozen kidneys using the Nucleospin RNA Midi kit [Macherey Nagel; 740962.20] as per manufacturers’ directions. RNA was assessed for quantity and quality using the TapeStation 4200 RNA ScreenTape kit [Agilent; 5067-5576], which showed all RNA used had a RIN of >8. Bulk RNA-seq was performed using the NEBNext Ultra II RNA Library Kit for Illumina [NEB; E7760] and NEBNext rRNA Depletion Kit (Human/Mouse/Rat) [NEB; E6310] as described in the manufacturers’ protocol, with 100ng of total RNA as input.
Refer to extract protocol for each individual file
Single cell libraries prepared using 10x 3’ v2&v3 kits following the manufacturers protocol. Bulk libraries were prepared using the NEBNext ultra II directional RNA library prep kit for Illumina following the manufacturers protocol. For the bulk libraries, rRNA was removed using either poly(A)+ selection using the NEBNext Poly(A) mRNA magnetic isolation module [E7490], or the NEBNext rRNA depletion kit (human/mouse/rat) kit [E6310].

value: Balb/c

value: Male

value: Bulk RNA-seq of whole kidney

value: bulk RNA-seq


title: JH4_parental_NT_a_rep1

description: aligned to AJ851868/mm9 hybrid genome

value: v-Abl transformed pro-B cell line

value: Emu-Bcl2, RAG2 deficient

tag: treatment protocol

value: RAG2 nucleofected, 3 uM STI-571 treated for 4 days

tag: growth protocol


title: JH4_CTCFdegron_IAA_a_rep1

description: aligned to AJ851868/mm9 hybrid genome

value: CTCFdegron, Emu-Bcl2, RAG2 deficient

value: RAG2 nucleofected,  500 uM IAA 6h, 3 uM STI-571 with 500uM IAA treated for 4 days


title: MJ12_Bulk


title: MJ14_Bulk


title: siNT1_ATAC-seq rep3


title: siSM2_ATAC-seq  rep3

description: Smarca2 knockdown


title: 235-1-1

value: Inflamed-before


title: 234-2-4

value: Uninflamed-after


title: JH4_Rad21degron_deIGCR1_IAA_b_rep1

description: aligned to AJ851868/mm9 hybrid genome

value: v- Abl transformed pro-B cell line

value: IGCR1deleted, Rad21degron, Emu-Bcl2, RAG2 deficient


title: 234-2-2


title: JH4_parental_NT_b_rep1

description: aligned to AJ851868/mm9 hybrid genome

value: RAG2nucleofected, 3 uM STI-571 treated for 4 days


title: 234-2-3


title: siGLI1_ATAC-seq  rep2

description: Gli1 knockdown


title: MJ08_SN_FANS_3×500g

value: Single-nuclei isolation, FANS, three washes and centrifugation speed of 500g

value: snRNA-seq, 10x v2 chemistry


title: 234-2-1


title: JH4_Rad21degron_deIGCR1_NT_b_rep1

description: aligned to AJ851868/mm9 hybrid genome

value: IGCR1deleted,Rad21degron, Emu-Bcl2, RAG2 deficient


title: 234-1-4


title: siNT2_ATAC-seq  rep2

value: non-targeting


title: JH4_CTCFdegron_IAA_b_rep1

description: aligned to AJ851868/mm9 hybrid genome


title: JH4_Rad21degron_IAA_a_rep1

description: aligned to AJ851868/mm9 hybrid genome

value: Rad21degron, Emu-Bcl2, RAG2 deficient


title: siNT1_ATAC-seq rep2


title: MJ05_SC_ColdDiss_Fresh

value: Balb/c

value: Male

value: Cold tissue dissociation, fresh suspension

value: scRNA-seq, 10x v2 chemistry


title: siGLI1_ATAC-seq  rep1

description: Gli1 knockdown


title: MJ04_SC_ColdDiss_Fresh


title: siNT2_ATAC-seq  rep1


title: MJ07_SN_FANS_3×500g


title: MJ03_SC_ColdDiss_Fresh


title: JH4_Rad21degron_NT_a_rep1

description: aligned to AJ851868/mm9 hybrid genome

value: RAG2 nucleofected, 3 uM STI-571 treated for 4 days


title: JH4_Rad21degron_IAA_b_rep1

description: aligned to AJ851868/mm9 hybrid genome


title: siSM2_ATAC-seq  rep1

description: Smarca2 knockdown


title: BG22_BulkSeq_WarmDiss_Fresh

extract_protocol:
Warm tissue dissociation  Kidneys were dissociated using the Multi-tissue dissociation kit 2 from Miltenyi Biotec [130-110-203] as per manufacturers’ instruction, with minor variations. Once the weight of the kidney was determined, the kidney was quartered and placed into a gentleMACS C-tube [Miltenyi Biotech; 130-096-334] containing the enzyme mix described in the kit’s protocol. The tube was centrifuged briefly, then placed onto the gentleMACS octo dissociator (Miltenyi Biotech), and the 37C_Multi_E program was run after attaching the heating elements. Following completion of the program, the tube was briefly centrifuged.   The homogenate was filtered through a 70µm cell strainer [Greiner; 54207] into a 50mL centrifuge tube [Greiner; 227270], the strainer was then rinsed with 15mL of PBS. The cell suspension was centrifuged at 400g for 10 minutes; once complete, the supernatant was removed and the pellet was resuspended in 5mL of PBS+0.04% BSA [Sigma; A7638]. The cell suspension was then filtered through a 40µm strainer [Greiner; 542040], which was subsequently rinsed with 2mL of PBS+0.04% BSA. The cells were again centrifuged at 400g for 10 minutes. The supernatant was then removed and the pellet was resuspended in 5mL of PBS+0.04% BSA. Cell count and viability was estimated using the Countess II FL (ThermoFisher) and the ReadyProbes Blue/Red kit [Invitrogen; R37610]. Bulk RNA-seq was performed using the NEBNext Ultra II RNA Library Kit for Illumina [NEB; E7760] and NEBNext rRNA Depletion Kit (Human/Mouse/Rat) [NEB; E6310] as described in the manufacturers’ protocol, with 100ng of total RNA as input.
Refer to extract protocol for each individual file
Single cell libraries prepared using 10x 3’ v2&v3 kits following the manufacturers protocol. Bulk libraries were prepared using the NEBNext ultra II directional RNA library prep kit for Illumina following the manufacturers protocol. For the bulk libraries, rRNA was removed using either poly(A)+ selection using the NEBNext Poly(A) mRNA magnetic isolation module [E7490], or the NEBNext rRNA depletion kit (human/mouse/rat) kit [E6310].

value: Bulk RNA-seq of warm-dissociated fresh cell suspension

value: bulk RNA-seq


title: siSM2_ATAC-seq  rep2

description: Smarca2 knockdown


title: MJ06_SN_FANS_3×500g


title: TT-seq_MJ-20-5_HCT-116_MED14-dTAG_2h_dTAGV-1_B

description: cross-compare only to MJ-20-5 samples!

data_processing:
Library strategy: TT-seq
alignment: STAR v2.3.0 to a fused index of hg38_ERCC; later filtered with 'samtools view -q 7 -f2'
spike-in_counting: reads aligning to ERCC spike-ins were counted with HTSeq
Further data processing was carried out using the R/Bioconductor environment, essentially as described (PMID: 27257258).
Genome_build: hg38
Supplementary_files_format_and_content: bigWig generation: strand-specific signal tracks were computed with 'bedtools genomeCoverageBed -bg -strand' followed by UCSC 'bedGraphToBigWig' and normalized based on signal coverage in 25kb windows at the end of very long (> 175kb) genes, which are too long to be affected by the applied short-term MED14 degradation (limited by Pol II elongation velocity).

source_name: HCT-116 colon carcinoma cell line

growth_protocol: KBM7 cells were grown in IMDM medium supplemented with 10% FBS and 1% Pen/Strep. HCT-116 cells were grown in RPMI medium supplemented with 10% FBS and 1% Pen/Strep

extract_protocol:
TT-seq libraries were generated as previously described (PMID: 27257258). Cells were harvested by 3000g for 2min and total RNA extracted using Qiazol. RNA spike-ins were added and RNA was sonicated to generate fragments of <1.5 kb. 4sU-labeled RNA was purified using streptavidin beads (Miltenyi Biotec) before size-selection and library preparation.
NuGEN Ovation Universal RNA-Seq System, starting with 100 ng RNA inputs.

treatment_protocol:
150-200mio KBM7 MED14-dTAG cells were treated for 0.5-2h with 500nM dTAG7, 500nM NVP2, or DMSO. 500 µM 4-thio-Uridine (4sU) were added from DMSO stocks for the last 5min to label newly synthesized RNA. 5x20mio HCT-116 MED14-dTAG cells grown in 15cm dishes were treated for 1-2h with 500nM dTAGV-1 or DMSO and labeled with 500µM 4-thio-Uridine (4sU) from DMSO stocks for the last 10min to label newly synthesized RNA.
150-200mio KBM7 MED14-dTAG cells were treated for 0.5-2h with 500nM dTAG7, 500nM NVP2, or DMSO. 500 ÂµM 4-thio-Uridine (4sU) were added from DMSO stocks for the last 5min to label newly synthesized RNA. 5x20mio HCT-116 MED14-dTAG cells grown in 15cm dishes were treated for 1-2h with 500nM dTAGV-1 or DMSO and labeled with 500ÂµM 4-thio-Uridine (4sU) from DMSO stocks for the last 10min to label newly synthesized RNA.

value: 2h 500nM dTAGV-1

tag: experiment id


title: TT-seq_MJ-20-5_HCT-116_MED14-dTAG_2h_dTAGV-1_A

description: cross-compare only to MJ-20-5 samples!


title: siNT1_ATAC-seq rep1


title: TT-seq_MJ-20-5_HCT-116_MED14-dTAG_2h_DMSO_B

description: cross-compare only to MJ-20-5 samples!

value: 2h DMSO


title: JH4_Rad21degron_NT_b_rep1

description: aligned to AJ851868/mm9 hybrid genome

value: v- Abl transformed pro-B cell line


title: BG21_BulkSeq_ColdDiss_Fresh

extract_protocol:
Cold tissue dissociation  Kidneys were dissociated using a modified version of the published protocol described in (Adam, Potter, & Potter, 2017). Based on the weight, in a pre-cooled Miltenyi C-tube, a protease solution (5mM CaCl2 [Invitrogen; AM9530G], 10mg/mL B. Licheniformis protease [Sigma; P5380], 125U/mL DNase I [Sigma; D5025], 1xDPBS) was prepared for each kidney.  The kidneys were then minced on ice into a smooth paste using a scalpel. The minced kidney was transferred into 4-6mL of the protease solution (dependent on weight) and triturated using a 1mL pipette for 15 seconds every two minutes for a total of eight minutes.  Following trituration, the C-tubes were placed onto a Miltenyi gentleMACS octo dissociator in a cool room (4°C), and the m_brain_03 program was run twice in succession. Once complete, the samples were triturated for 15 seconds every two minutes on ice for an additional 16 minutes using a 1mL pipette. 10µL of each sample was then loaded into a haemocytometer to assess whether tissue dissociation was complete. The dissociated cells were transferred to a 15mL centrifuge tube and 3mL of ice-cold PBS+10%FBS [Gibco; A3160401] was added.   The cell suspension was centrifuged at 1200g for five minutes at 4°C. The supernatant was removed and the pellet was resuspended in 2mL of PBS+10%FBS. The cells were then filtered through a 70µm cell strainer, which was subsequently rinsed with 2mL of PBS+0.01% BSA. The cells were then centrifuged again at 1200g for five minutes at 4°C followed by removal of the supernatant and resuspension of the pellet in 5mL of PBS+0.01%BSA. The cells were then filtered through a 40µm cell strainer, which was subsequently rinsed with 2mL of PBS+0.01% BSA. The cells were again centrifuged at 1200g for five minutes at 4°C followed by removal of the supernatant and resuspension of the cells in 5mL of PBS+0.04%BSA. The cells were counted and checked for viability using the ReadyProbes Blue/Red Kit on the Countess II FL. Bulk RNA-seq was performed using the NEBNext Ultra II RNA Library Kit for Illumina [NEB; E7760] and NEBNext rRNA Depletion Kit (Human/Mouse/Rat) [NEB; E6310] as described in the manufacturers’ protocol, with 100ng of total RNA as input.
Refer to extract protocol for each individual file
Single cell libraries prepared using 10x 3’ v2&v3 kits following the manufacturers protocol. Bulk libraries were prepared using the NEBNext ultra II directional RNA library prep kit for Illumina following the manufacturers protocol. For the bulk libraries, rRNA was removed using either poly(A)+ selection using the NEBNext Poly(A) mRNA magnetic isolation module [E7490], or the NEBNext rRNA depletion kit (human/mouse/rat) kit [E6310].

value: Bulk RNA-seq of cold-dissociated fresh cell suspension


title: BG20_BulkSeq_WarmDiss_Fresh


title: 234-1-3

extract_protocol: Gut biopsies were collected in RNAlater Stabilization Solution (Ambion, Applied Biosystems) and then frozen on dry ice. Samples were sent on dry ice to KFB Center of Excellence for Fluorescent Bioanalytics (Regensburg, Germany) for extracting materials.

value: Inflamed-before


title: 233-2-4

value: Uninflamed-after


title: 234-1-1

value: Uninflamed-before


title: 233-2-1


title: 234-1-2


title: siGLI1_RNA-seq  rep3

description:
15-064-4
Gli1 knockdown

data_processing:
Base calling was performed with the Illumina bcl2fastq
Sequences were trimmed with TrimGalore (v0.4.5), aligned to hg19 (NCBI build 37) using bowtie2 (v2.3.3.1) with maximum fragment length 2000.  FeatureCounts (Subread v1.6.4) was used to count reads and summarize then Deseq2 (v2.11.40.6 in Galaxy) was used to determine differentially expressed genes.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include normalized values for each gene per sample

extract_protocol:
mRNA was isolated from cultured cells following the protocol in the Qiagen RNeasy kit including the DNase I step.
mRNA version 2 library prep kit (Illumina)

treatment_protocol: siRNA knockdowns of Gli1 were incubated for 72 hours in standard cell culture conditions.


title: 233-2-3


title: 233-2-2-repeat


title: 233-2-2


title: siNT2_RNA-seq  rep3

description:
15-064-3
Cntrl2


title: BG18_BulkSeq_WarmDiss_Fresh


title: siGLI1_RNA-seq  rep2

description:
15-063-4
Gli1 knockdown


title: TT-seq_MJ-20-5_HCT-116_MED14-dTAG_1h_DMSO_B

description: cross-compare only to MJ-20-5 samples!

value: 1h DMSO


title: TT-seq_MJ-20-5_HCT-116_MED14-dTAG_1h_dTAGV-1_B

description: cross-compare only to MJ-20-5 samples!

value: 1h 500nM dTAGV-1


title: TT-seq_MJ-20-5_HCT-116_MED14-dTAG_1h_dTAGV-1_A

description: cross-compare only to MJ-20-5 samples!


title: siNT2_RNA-seq  rep2

description:
15-063-3
Cntrl2

value: non-targeting


title: BG17_BulkSeq_ColdDiss_Fresh


title: BG57_SC_WarmDiss_Methanol

extract_protocol:
Warm tissue dissociation  Kidneys were dissociated using the Multi-tissue dissociation kit 2 from Miltenyi Biotec [130-110-203] as per manufacturers’ instruction, with minor variations. Once the weight of the kidney was determined, the kidney was quartered and placed into a gentleMACS C-tube [Miltenyi Biotech; 130-096-334] containing the enzyme mix described in the kit’s protocol. The tube was centrifuged briefly, then placed onto the gentleMACS octo dissociator (Miltenyi Biotech), and the 37C_Multi_E program was run after attaching the heating elements. Following completion of the program, the tube was briefly centrifuged.   The homogenate was filtered through a 70µm cell strainer [Greiner; 54207] into a 50mL centrifuge tube [Greiner; 227270], the strainer was then rinsed with 15mL of PBS. The cell suspension was centrifuged at 400g for 10 minutes; once complete, the supernatant was removed and the pellet was resuspended in 5mL of PBS+0.04% BSA [Sigma; A7638]. The cell suspension was then filtered through a 40µm strainer [Greiner; 542040], which was subsequently rinsed with 2mL of PBS+0.04% BSA. The cells were again centrifuged at 400g for 10 minutes. The supernatant was then removed and the pellet was resuspended in 5mL of PBS+0.04% BSA. Cell count and viability was estimated using the Countess II FL (ThermoFisher) and the ReadyProbes Blue/Red kit [Invitrogen; R37610].   Methanol fixation  Fixing  The methanol-fixation protocol was based on (Chen, Cheung, Shi, Zhou, & Lu, 2018). After tissue dissociation, the cells were concentrated to approximately 5x106 cells/mL by centrifuging at 1000g for 10 minutes. 200µL of the cell suspensions were aliquoted into 2mL cryovials resting on ice. 800µL of 100% methanol [Sigma; 494437] (chilled at -20°C) was then added dropwise to each sample while gently stirring the cells to prevent clumping. The cryovials were stored at -20°C for 30 minutes, then directly transferred to -80°C (no gradient cooling).  Rehydrating  Cryovials of methanol fixed cells were removed from -80°C and placed on ice to equilibrate to 4°C (approximately 10 minutes). The cells were then transferred to a 1.5mL centrifuge tube and centrifuged at 1000g for five minutes at 4°C. The supernatant was discarded and the pellet was resuspended in a small volume of SSC cocktail (3xSSC [Sigma; S0902], 0.04% BSA, 40mM DTT [Sigma; 43816], 0.5U/mL RNasin plus [Promega; N2615]) to reach a concentration of approximately 2000cells/µL. The cells were then filtered through a pre-wetted (with 1mL of nuclease-free water) 40µm pluristrainer mini filter [PluriSelect; 43-10040]. The cells were counted using the ReadyProbes Blue/Red Kit on the Countess II, then adjusted to 2000cells/µL based on the count. The cells were loaded onto a 10x chip (A/B depending on version used) at a volume that dilutes the SSC to 0.125x to prevent reverse transcription inhibition. All single cell libraries were constructed in biological triplicate using the 10x Chromium 3’ workflow as per manufacturers’ directions.
Refer to extract protocol for each individual file
Single cell libraries prepared using 10x 3’ v2&v3 kits following the manufacturers protocol. Bulk libraries were prepared using the NEBNext ultra II directional RNA library prep kit for Illumina following the manufacturers protocol. For the bulk libraries, rRNA was removed using either poly(A)+ selection using the NEBNext Poly(A) mRNA magnetic isolation module [E7490], or the NEBNext rRNA depletion kit (human/mouse/rat) kit [E6310].

value: Warm tissue dissociation, methanol-fixed suspension


title: TT-seq_MJ-20-5_HCT-116_MED14-dTAG_2h_DMSO_A

description: cross-compare only to MJ-20-5 samples!


title: BG19_BulkSeq_ColdDiss_Fresh

value: Male


title: 233-1-4


title: TT-seq_MJ-19-29_MED14-dTAG_2h_dTAG7_30min_NVP2_B

description: cross-compare only to MJ-19-29 samples

data_processing:
library strategy: TT-seq
alignment: STAR v2.3.0 to a fused index of hg38_ERCC; later filtered with 'samtools view -q 7 -f2'
spike-in_counting: reads aligning to ERCC spike-ins were counted with HTSeq
Further data processing was carried out using the R/Bioconductor environment, essentially as described (PMID: 27257258).
Genome_build: hg38
Supplementary_files_format_and_content: bigWig generation: unnormalized, strand-specific signal tracks were computed with 'bedtools genomeCoverageBed -bg -strand' followed by UCSC 'bedGraphToBigWig'

source_name: KBM7_MED14-dTAG_2h 500nM dTAG7 and last 30min 500nM NVP2

treatment_protocol:
150-200mio KBM7 MED14-dTAG cells were treated for 0.5-2h with 500nM dTAG7, 500nM NVP2, or DMSO. 500 µM 4-thio-Uridine (4sU) were added from DMSO stocks for the last 5min to label newly synthesized RNA. 5x20mio HCT-116 MED14-dTAG cells grown in 15cm dishes were treated for 1-2h with 500nM dTAGV-1 or DMSO and labeled with 500µM 4-thio-Uridine (4sU) from DMSO stocks for the last 10min to label newly synthesized RNA.
150-200mio cells were treated for 0.5-2h with 500nM dTAG7, 500nM NVP2, or DMSO. 500 ÂµM 4-thio-Uridine (4sU) were added from DMSO stocks for the last 5min to label newly synthesized RNA.

value: chronic myeloid leukemia cell line

tag: genotype/variation


title: siNT2_RNA-seq  rep1

description:
15-060-3
Cntrl2