title: CropSeq NTN, Sample 4

city: Hamburg

first: Malte

last: Hellmig

state: Hamburg

address: Martinistraße 52

department: III. Department of Medicine

institute: University Medical Center Hamburg-Eppendorf

description: 10X Genomics scRNA-seq using Chromium Single Cell 3' v6

data_processing:
10x Genomics raw sequencing data were processed using CellRanger software (version 3.1, 10x Genomics, Pleasanton, CA).
Assembly: mm10
Supplementary files format and content: Cellranger output: Tab seperated values files and Matrix table with raw gene counts

source_name: kidney

extract_protocol:
Kidneys were incubated at 37 °C for 40 min in RPMI 1640 Medium (Thermo Fisher) containing 10% heat inactivated FBS, 1% P/S, 0.4 µg/ml Collagenase D (Roche, Basel, Switzerland) and 0.2 µg/ml DNAse (Roche). Subsequently, tissue was dissociated using the GentleMACS Dissociator and C-Tubes (both Miltenyi Biotec) and leukocytes were isolated using a gradient centrifugation with 37 % Percoll (GE Healthcare). After the centrifugation, cells were washed with DPBS (Gibco). CD4+ T cells were enriched using the EasySep Mouse CD4+ T cell Isolation Kit according to manufacturer’s instructions (Stemcell Technologies, Vancouver, Canada). Enriched cells were sorted by Fluorescence-activated cell sorting (FACS) for CD4+ T cells, GFP signal and eBFP2 signal.
All steps were performed according to standard protocol of the Chromium Single Cell 3' v3 kit

value: kidney

value: T cells

value: crescentic glomerulonephritis


title: blood sample from stimulated rat at 2 days post status epilepticus, biological replicate 2

status: Public on Jun 14 2024

type: RNA

city: Warsaw

first: Kinga

last: Szydlowska

state: Select State

address: Pasteura 3

country: Poland

institute: Nencki Institute of Experimental Biolo

description: Gene expression data from blood of adult rats after amygdala stimulation

hyb_protocol: Hybridization was done for 20 hours in +48 °C (AF-900454 GeneChip Hybridization Control Kit, Affymetrix). It was followed by the wash and stain protocol (AF-901910 FlashTagTM Biotin HSR RNA Labeling Kit, Affymetrix). The whole procedure was performed using GeneAtlas™ instrument from Affymetrix (#00-0393).

scan_protocol: The whole procedure was performed using GeneAtlas™ instrument from Affymetrix.

data_processing:
Data analysis of the miRNA 4.1 Array Strips identifying up- and down-regulated miRNAs was performed with RStudio (R v. 3.3.2) using Bioconductor (Gentleman et al. 2004, R_Core_Team 2012), oligo and limma (G 2005) packages. The microarray data were normalized with the Robust Multi-array Average (RMA) algorithm (oligo package version 1.22.0) (Carvalho and Irizarry 2010). For the expression analysis, the calculated p-values were based on moderated t-statistics. Furthermore, the Benjamini and Hochberg multiple testing adjustment method was applied to the p-values (FDR – False Discovery Rate). A one-way ANOVA was used to establish  miRNA with differential levels between the tested groups.  The correlation, unsupervised clustering and PCA (principal component analyses) were performed in the RStudio (R version 3.3.2) (R_Core_Team 2012). PCA was performed on microarray probes using package stats and the lattice package (Sakar D 2008). For the heatmap clustering of miRNAs with significantly different level (p < 0.05), miRNAs were ordered with the clustering complete-linkage method, together with the Pearson correlation distance measure. The heatmap diagrams were generated with the gplots package (Warnes GR 2012). Pearson correlation test was used to analyze the correlations between miRNAs with significantly different level (p < 0.05) between stimulated and sham-operated control animals. The fuzzy c-means algorithm implemented in the Mfuzz package (Kumar & E Futschik, 2007) was used to perform clusterization on all probes.  Data analysis of the miRNA 4.1 Array Strips identifying up- and down-regulated miRNAs was performed with RStudio (R v. 3.3.2) using Bioconductor (Gentleman et al. 2004, R_Core_Team 2012), oligo and limma (G 2005) packages. The microarray data were normalized with the Robust Multi-array Average (RMA) algorithm (oligo package version 1.22.0) (Carvalho and Irizarry 2010). For the expression analysis, the calculated p-values were based on moderated t-statistics. Furthermore, the Benjamini and Hochberg multiple testing adjustment method was applied to the p-values (FDR – False Discovery Rate). A one-way ANOVA was used to establish  miRNA with differential levels between the tested groups.  The correlation, unsupervised clustering and PCA (principal component analyses) were performed in the RStudio (R version 3.3.2) (R_Core_Team 2012). PCA was performed on microarray probes using package stats and the lattice package (Sakar D 2008). For the heatmap clustering of miRNAs with significantly different level (p < 0.05), miRNAs were ordered with the clustering complete-linkage method, together with the Pearson correlation distance measure. The heatmap diagrams were generated with the gplots package (Warnes GR 2012). Pearson correlation test was used to analyze the correlations between miRNAs with significantly different level (p < 0.05) between stimulated and sham-operated control animals. The fuzzy c-means algorithm implemented in the Mfuzz package (Kumar & E Futschik, 2007) was used to perform clusterization on all probes.
gc-rma signal

label: biotin

molecule: total RNA

organism: Rattus norvegicus

source_name: adult, male rat

label_protocol: miRNA level was profiled using miRNA 4.1 Array Strip (#902404, Santa Clara, CA, Affymetrix). Procedure was performed according to manufacturer’s recommendations. 8 µl of RNA was used to perform poly-A-tailing reaction (AF-902134 GA HWS Kit for miRNA Arrays, Affymetrix).

growth_protocol: Adult, male Sprague-Dawley rats (270-320 g) from Mossakowski Medical Research Centre Polish Academy of Sciences, in Warsaw (Poland) were used in this study. Rats were housed under controlled conditions (24 °C, humidity 50 - 60 %, 12/12 h light-dark cycle) with food and water available ad libitum. Animals were housed in pairs. Environment was enriched by using various toys (wooden or plastic balls, wooden bridges, etc.) and snacks, which were changed every week. Starting four weeks before electrical stimulation rats were subjected to regular handling every other day for 10 minutes.

extract_protocol: Blood drawn from rat tail vein was drawn at 3 time points and snap frozen. miRNA was isolated from 500 µl of blood using miRNeasy Kit (#217084, Qiagen). The whole miRNA isolation procedure was performed as described by the kit manufacturer. In the end of the procedure RNA was eluted to 100 µl of RNAse free water, aliquoted and stored in -80°C.

treatment_protocol: The amygdala stimulation model of temporal lobe epilepsy was used in this study as previously described with some modifications (Guzik-Kornacka et al, 2011; Nissinen et al., 2000). Surgery was performed under isoflurane anesthesia (2-2.5 % in 100 % O2) followed by the injection of butorphanol (Butomidor, Richter Pharma AG, Wells, Austria; 0.5 mg/kg i.p.) for analgesia. A stimulating and recording bipolar wire electrode (Plastic One Inc., Roanoke, VA, # E363-3-2WT-SPC) was implanted into the left lateral nucleus of the amygdala 3.6 mm posterior and 5.0 mm lateral to bregma, 6.5 mm ventral to the surface of the brain. A stainless steel screw electrode (Plastic One Inc., Roanoke, VA, #E363/20) was implanted contralaterally into the skull over the right frontal cortex (3.0 mm anterior and 2.0 mm lateral to bregma) as a surface EEG recording electrode. Two stainless steel screw electrodes were placed bilaterally over the cerebellum (10.0 mm posterior and 2.0 mm lateral to bregma) as grounding and reference electrodes. The contacts of all electrodes were placed in a multi-channel electrode pedestal (Plastic One Inc., Roanoke, VA, #MS363) which was attached to the skull with dental acrylate (Duracryl Plus). After two weeks of recovery, animals were electrically stimulated via the intra-amygdala electrode to evoke status epilepticus. Stimulation consisted of a 100-ms train of 1-ms biphasic square-wave pulses (400 μA peak to peak) delivered at 60 Hz every 0.5 s for 30 min. If the animal did not enter status epilepticus, stimulation was continued for an additional 10 min. The status epilepticus was stopped 1 h after stimulation via an intraperitoneal injection of diazepam (20 mg/kg). If the first dose of diazepam did not suppress status epilepticus , the animal received subsequent doses of diazepam at   5 mg/kg. Time matched control animals had electrodes implanted but did not receive electrical stimulation (sham animals).

tag: tissue

value: whole blood

tag: blood collection tube coating

value: tubes coated with 1 mg K2EDTA #363706 BD Vacutainer

tag: strain

value: Sprague Dawley


title: CropSeq NTN, Sample 5

type: SRA

country: Germany

description: 10X Genomics scRNA-seq using Chromium Single Cell 3' v7

library_source: transcriptomic single cell

molecule: polyA RNA

organism: Mus musculus

tag: cell type

tag: treatment


title: Donor 3, B cells, GSK3i, Nil

city: Philadelphia

first: Diego

middle: A

last: Espinoza

state: PA

address: 3400 Civic Center Boulevard

country: USA

institute: University of Pennsylvania

description: Donor 3 Nil GSK3i

library_source: transcriptomic

data_processing:
Data was analyzed using online platform Basepair (Link: https://www.basepairtech.com/)
Sequence reads were trimmed and mapped to hg19 using STAR algorithm (default settings on the platform)
Assembly: hg19
Supplementary files format and content: tab-delimited text file includes raw count values for each Sample

organism: Homo sapiens

source_name: Blood

growth_protocol: B cells were maintained in X-vivo 10 Medium supplement with Glutamine and antibiotics in humidified atmosphere with 5% CO2 at 37C

extract_protocol:
RNA was harvested using Rneasy micro plus kit (Qiagen). 200ng of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using SMARTER mRNA-Seq Library Prep Kit following manufacturer's protocols.

treatment_protocol: Vehicle (DMSO), BTK inhibitor (1uM) or GSK3 inhibitor (1uM) were added to B cells 30 min. B cells treated with Vehicle or BTK inhibitor was either left alone or stimulated by anti-BCR for 24hrs.

tag: individual

value: Donor 3

value: Blood

value: B cells

tag: disease state

value: Healthy

tag: time


title: blood sample from control rat (CTRL vs. SE) at 7 days post status epilepticus, biological replicate 2


title: Donor 2, B cells, Vehicle, anti-BCR

description: Donor 2 aBCR Vehicle

value: Donor 2

value: Anti-BCR Vehicle


title: Input of ChIP_CpATAP_HeLa_02

city: Bethesda

first: Songjoon

last: Baek

state: MD

address: 41 Library Drive

department: CCR

institute: NCI / NIH

library_source: genomic

data_processing:
Base-calling with Illumina run time analysis software 1.12.4.2
Alignment to hg38 using bowtie2 (ver 2.3.4.1).  Narrow peaks were called using the MACS2 (version 2.1.1.20160309)  callpeak function using the sample’s respective input dataset.
The replicate concordant peaks among replicates were combined to get the highly reproducible peaks using the IDR method.
Assembly: hg38
Supplementary files format and content: narrowPeak

molecule: genomic DNA

source_name: HeLa

extract_protocol:
Samples were digested to mononucleosomes (~20 min MNase) and ChIP'ed using anti- CENP-A or S-tag antibodies. Purified DNA fragments were sent for sequencing.
Sequencing libraries were generated using Illumina TruSeq V3 protocol and subject to 101 bp paired-end sequencing on Illumina NextSeq2000.

tag: cell line

value: HeLa

value: Transfected HeLa cells

tag: chip antibody


title: knockout rep4

city: BOSTON

first: CHANG

last: CAO

state: MA

address: 185 CAMBRIDGE ST.

department: CENTER FOR GENOMIC MEDICINE

institute: Mass General Brigham

data_processing:
Image analysis and base calling were conducted by the HiSeq Control Software. Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification.
Reads were trimmed by trimmomatic v0.36 and high-quality reads were aligned to mouse reference genome (GRCm38.p6) using STAR 2.5.2b.
The reads counts were calculated by featureCounts software from the Subread package v1.5.2.
Assembly: GRCm38.p6
Supplementary files format and content: Matrix table with normalized gene counts for every gene and every sample

source_name: placenta

extract_protocol:
RNA was extracted from placenta using Rneasy plus mini kit (Qiagen, cat. no. 73404).
NEBNext Ultra RNA Library Prep Kit for Illumina (cat. no. E7770) was used for the construction of sequencing libraries following manufacturer’s instructions.

value: placenta

tag: genotype

value: KO

tag: Sex

value: female

value: Embryonic day 15.5


title: imDCs,Meso_3

city: Guiyang

first: Ｗang

last: Yun

state: Guizhou province

address: School of Basic Medicine

country: China

institute: Guizhou Medical University

data_processing:
DNBSEQ-T7
clean reads were compared to the reference genome sequence using HISAT
Bowtie2 was used to compare clean reads to reference gene sequences to obtain comparison results
Supplementary files format and content: tab-deliminted text file includes raw couts for each sample

source_name: immature dendritic cells

growth_protocol: Male C57BL/6 mice (6 weeks old) were sacrificed after anesthesia, femur and tibia were dissected, bone marrow cells were washed with PBS solution, and red blood cells were removed from cell suspension with red blood cell lysate. Complete media (containing 10% FBS, 20 ng/mL of rmGM-CSF and 10 ng/mL of rmIL-4) were added to re-suspension cell suspensions, and cell suspensions were subpackaged and cultured in six-well plates, and the liquid was changed every other day and half, and immature DCs was obtained on day 6.

extract_protocol:
ImDCs cultured on tissue culture plates（Control）and the surfaces of HA dishes (Nano and Meso) for 24 h were taken into the grinding and crushing tube with corresponding number. Add 600μL Buffer RLT Plus, homogenize the grinding machine, and let it stand at room temperature for 5 min; At room temperature, centrifuge at 12,000×g for 3 min; All supernatants were transferred to AllPrep DNA spin column; Centrifuge at room temperature of 10,000×g for 30 s, and the liquid in the collection tube was purified and collected for RNA
The DNBs obtained were added to the mesh holes on the chip using high-density DNA nanochip technology, and sequenced by combined probe anchoring polymerization (cPAS)

treatment_protocol: High purity imDCs was obtained after purification by CD11c+ magnetic beads, and cultuerd ontissue culture plates（Control）and the surfaces of HA dishes (Nano and Meso) for 24 h．

value: immature dendritic cells

value: Meso

tag: time point


title: Donor 2, B cells, GSK3i, Nil

description: Donor 2 Nil GSK3i


title: CpATAP ChIP in transfected HeLa rep #2

value: Millipore Cat #MAC115


title: imDCs,Meso_2


title: blood sample from stimulated rat at 2 days post status epilepticus, biological replicate 6

value: whole blood

value: tubes coated with 1 mg K2EDTA #363706 BD Vacutainer


title: knockout rep3


title: blood sample from stimulated rat at 2 days post status epilepticus, biological replicate 1

value: Sprague Dawley


title: Donor 1, B cells, BTKi, anti-BCR

description: Donor 1 aBCR BTKi

value: Donor 1

value: Blood

value: Anti-BCR BTKi


title: Donor 1, B cells, GSK3i, Nil

description: Donor 1 Nil GSK3i

value: B cells

value: Healthy


title: blood sample from control rat (CTRL vs. SE) at 2 days post status epilepticus, biological replicate 7


title: CpATAP ChIP in transfected HeLa rep #1

value: HeLa

value: Millipore Cat #MAC113


title: imDCs,Meso_1


title: Input of ChIP_CpATAP_HeLa_01

value: Transfected HeLa cells


title: blood sample from control rat (CTRL vs. SE) at 2 days post status epilepticus, biological replicate 6


title: Donor 3, B cells, BTKi, Nil

description: Donor 3 Nil BTKi

value: Donor 3

value: BTKi


title: imDCs,Nano_3

value: Nano


title: blood sample from control rat (CTRL vs. SE) at 2 days post status epilepticus, biological replicate 5


title: imDCs,Nano_2

value: immature dendritic cells


title: nCpA ChIP in HeLaCont rep #2

value: HeLa cells

value: Custom Rb CENP-A


title: Donor 2, B cells, BTKi, Nil

description: Donor 2 Nil BTKi

value: Donor 2


title: Input of ChIP_nCpA_HeLaCont_02


title: blood sample from control rat (CTRL vs. SE) at 2 days post status epilepticus, biological replicate 4

value: whole blood

value: tubes coated with 1 mg K2EDTA #363706 BD Vacutainer


title: Donor 1, B cells, BTKi, Nil

description: Donor 1 Nil BTKi

value: Donor 1


title: imDCs,Nano_1


title: nCpA ChIP in HeLaCont rep #1


title: blood sample from control rat (CTRL vs. SE) at 2 days post status epilepticus, biological replicate 3

value: Sprague Dawley


title: imDCs,Control_3


title: Donor 3, B cells, Vehicle, Nil

description: Donor 3 Nil Vehicle

value: Blood

value: Vehicle


title: Input of ChIP_nCpA_HeLaCont_01


title: Donor 2, B cells, Vehicle, Nil

description: Donor 2 Nil Vehicle

value: B cells

value: Healthy


title: blood sample from control rat (CTRL vs. SE) at 2 days post status epilepticus, biological replicate 2


title: Beads only IP

value: HeLa


title: imDCs,Control_2


title: blood sample from control rat (CTRL vs. SE) at 2 days post status epilepticus, biological replicate 1


title: Mouse_Brain_Buffer_only_PFA_rep2

status: Public on Jun 13 2024

city: La Jolla

first: Brian

last: Yee

state: CA

address: 9500 Gilman Dr

department: Health

institute: UCSD

data_processing:
remove adapter with Cutadapt
align to hg38 using STAR 2.4.0 (Homo sapiens) or mm10 using STAR 2.5.2 (Mus musculus)
SAILOR analysis to call C-to-U edits and keep only sites with score >0.5 and edit fraction <80%
FLARE analysis to call C-to-U edit clusters
Intersect the edit clusters from 3 replicates, which yields "*confident_peaks.bed"
Subtract STAMP confident clusters to Buffer only control, which yields "*cleaned_confident_peaks.bed"
Assembly: hg38/mm10
Supplementary files format and content: SAILOR step yields bed file: *0.5Score0.8Fraction.fastqTr.sorted.STARUnmapped.out.sorted.STARAligned.out.sorted.bam.combined.readfiltered.formatted.varfiltered.snpfiltered.ranked.bed
Supplementary files format and content: FLARE step yields .tsv file: "*merged_sorted_peaks.fdr_0.1.d_15.scored.tsv"
Supplementary files format and content: Intersection step yields .bed file:  "*confident_peaks.bed"
Supplementary files format and content: Subtraction step yields .bed file:  "*cleaned_confident_peaks.bed"

source_name: Brain

extract_protocol:
Direct-zol RNA Miniprep Kits, Zymo Research (Cat # R2052)
rRNA depleted RNA
NEBNext® Single Cell/Low Input RNA Library Prep Kit (NEB) / Ilumina TruSeq Stranded mRNA Library Prep (Cat #  20020594)

value: Female

value: Brain

value: whole brain section

tag: replicate


title: Donor 1, B cells, Vehicle, Nil

description: Donor 1 Nil Vehicle


title: Mouse_Brain_Buffer_only_PFA_rep3


title: Mouse_Brain_Buffer_only_PFA_rep1


title: Mouse_Brain_Enzyme_only_PFA_rep2

description:
Enzyme_only_PFA_confident_peaks.bed
Enzyme_only_PFA_cleaned_confident_peaks.bed


title: Mouse_Brain_RBFOX2_INSCRIBE_PFA_rep3

description:
RBFOX2_INSCRIBE_PFA_confident_peaks.bed
RBFOX2_INSCRIBE_PFA_cleaned_confident_peaks.bed


title: Mouse_Brain_Enzyme_only_PFA_rep3


title: Mouse_Brain_RBFOX2_INSCRIBE_PFA_rep2


title: Mouse_Brain_RBFOX2_INSCRIBE_PFA_rep1


title: Mouse_Brain_Enzyme_only_PFA_rep1


title: rem1a inoculated with Rhizobium NopL mutant at 24 hpi\, Repeat 2

city: Harbin

first: Chao

last: Ma

state: Hei Longjiang

address: 中国 哈尔滨 香坊区 长江路600号

department: College of Agriculture

institute: Northeast Agriculutral University

data_processing:
RSEM was used to quantify gene abundances. Essentially, differential expression analysis was performed using the DESeq2. DEGs with |log2FC|≧1 and FDR≤ 0.05 were considered to be significantly different expressed genes.
Assembly: Phytozome genome ID: 508 • NCBI taxonomy ID: 3847
Supplementary files format and content: Counts for RNA-seq of DN50 and Gmrem1a inoculated with Rhizobium

organism: Glycine max

source_name: root

growth_protocol: The soybean seedlings were grown in a greenhouse under a 16 h light / 8 h dark cycle at a temperature of 25 °C.

extract_protocol:
Total RNA extract by Trizol
Used by VAHTS Universal V10 RNA-seq Library Prep Kit for Illumina

treatment_protocol: These plants were inoculated with different strains of rhizobia, including the wild type HH103, derived mutants HH103ΩnopL and HH103ΩnodA and a mock control using 10mM MgSO4. The plants were inoculated with these rhizobia strains, and the roots were sampled,

value: root

value: 24 dpi


title: rem1a inoculated with Rhizobium HH103 at 24 hpi\, Repeat 1

address: 中国 哈尔滨 香坊区 长江路600号


title: rem1a inoculated with Rhizobium HH103 at 24 hpi\, Repeat 3

address: 中国 哈尔滨 香坊区 长江路600号


title: DN50 inoculated with Rhizobium NopL mutant at 24 hpi\, Repeat 2

address: 中国 哈尔滨 香坊区 长江路600号


title: DN50 inoculated with Rhizobium NopL mutant at 24 hpi\, Repeat 3

address: 中国 哈尔滨 香坊区 长江路600号


title: DN50 inoculated with Rhizobium NopL mutant at 24 hpi\, Repeat 1

address: 中国 哈尔滨 香坊区 长江路600号


title: imDCs,Control_1

value: immature dendritic cells


title: rem1a inoculated with Rhizobium NopL mutant at 24 hpi\, Repeat 3

address: 中国 哈尔滨 香坊区 长江路600号


title: DN50 inoculated with Rhizobium HH103 at 24 hpi\, Repeat 2

address: 中国 哈尔滨 香坊区 长江路600号


title: rem1a inoculated with Rhizobium HH103 at 24 hpi\, Repeat 2

address: 中国 哈尔滨 香坊区 长江路600号


title: InfL21

city: Münster

first: Robert

last: Peuß

address: Hüfferstrasse 1

department: Institute for Evolution and Biodiversiity

institute: University of Münster

data_processing:
Data processing was done as described in DOI: 10.1002/jezb.22933
Assembly: GCF_016920845.1
Supplementary files format and content: processed data file contains normalized count and fpkm values for each sample and gene

organism: Gasterosteus aculeatus

extract_protocol:
RNA was extracted from brain tissues and purified using the Promega SV RNA Extraction kit.
Libraries were prepared according to manufacturer's instructions using the TruSeq Stranded mRNA Prep Kit (Illumina)

tag: group

value: infected

value: late


title: InfL9


title: rem1a inoculated with Rhizobium NopL mutant at 24 hpi\, Repeat 1

address: 中国 哈尔滨 香坊区 长江路600号


title: ExpL15

value: exposed


title: InfL23


title: ExpL13


title: ExpL1


title: ExpL7


title: ConL20

value: control


title: ConL2


title: DN50 inoculated with Rhizobium HH103 at 24 hpi\, Repeat 3

address: 中国 哈尔滨 香坊区 长江路600号


title: ConL14


title: InfE47

value: early


title: InfE41


title: InfE27


title: DN50 inoculated with Rhizobium HH103 at 24 hpi\, Repeat 1

address: 中国 哈尔滨 香坊区 长江路600号


title: ExpE33


title: ExpE31


title: ConE40


title: ExpE37


title: ConE34


title: ExpE35


title: HEK293T_RBFOX_STAMP

description: Long-read in situ RBFOX2-APOBEC1 STAMP

data_processing:
Demultiplex primers with lima (v2.6.0)
Refine reads with Isoseq3 refine (v3.8.0)
Align reads using pbmm2 (v1.9.0)
Cluster reads with isoseq3 cluster (v3.8.0)
Filter for primary mapped reads with custom script
Identify gene editing using custom script
remove edits found in annotated SNP positions using custom script
Assembly: hg38
Supplementary files format and content: isoforms from clustering (.gff)
Supplementary files format and content: gene edits (.bed)

source_name: Kidney

extract_protocol:
Direct-zol RNA Miniprep Kits, Zymo Research (Cat # R2052)
PacBio Iso-seq SMRTbell prep kit 3.0 (PN: 102-396-000)

value: Kidney

value: immortalized cell line


title: 100pg_Enzyme_only_rep2

description:
100pg_Enzyme_only_confident_peaks.bed
100pg_Enzyme_only_cleaned_confident_peaks.bed


title: 100pg_Enzyme_only_rep1


title: 1ng_Enzyme_only_rep3

description:
1ng_Enzyme_only_confident_peaks.bed
1ng_Enzyme_only_cleaned_confident_peaks.bed


title: 100pg_Enzyme_only_rep3


title: InfE25


title: ConE28


title: HEK293T_APO_STAMP

description: Long-read in situ APOBEC1 only STAMP data


title: 1ng_Enzyme_only_rep1


title: 1ng_Enzyme_only_rep2


title: RNAseq_noMYCN_R3

city: Wuerzburg

first: Martin

last: Eilers

address: Am Hubland

department: Chair for Biochemistry and Molecular Biology

institute: University of Wuerzburg

description: polyA-RNAseq

data_processing:
Fastq files were generated Illumina’s BaseSpace platform and overall sequencing quality was analyzed with the FastQC script.
For CnR and ChIP-Rx, reads were aligned to hg19 and mm10 using Bowtie2 v2.3.5.1 with default parameters. For RNA, 4sU and 3'RNAseq reads were aligned to hg19 and mm10 using STAR v2.7.10a (2.7.6a for eCLIP). For 4sUseq reads mapping to rRNA, exons and blacklist regions were removed. All datasets (except the SIN3A and MNT CnR, which were normalized to sequencing depth) were normalized based on mm10 reads.
Following normalization, alignment files were converted to bigwig with deeptools bamCoverage v3.5.1. 3'RNAseq stranded bedgraphs were generated with a custom python script and bedgraphs were then converted to bigwig with bedGraphToBigWig v2.9. CLIPper v2.1.0 was used for eCLIP-seq peak calling. featureCounts v2.0.3 was used for RNAseq counts.
genome build/assembly: hg19, mm10
processed data files format and content: For ChIP-RX, eCLIP, 4sU-RNA and 3'RNA processed files are in bigwig format. All CnR processed files are also in bigwig format, except for SIN3A, MNT and MAX R2 files, which are in bedgraph format. The RNAseq processed file is a featureCounts output text file.

source_name: SHEP

growth_protocol:
Human neuroblastoma IMR-32 cells expressing doxycycline (Dox)-inducible shRNAs or expressing an endogenous AID-tagged EXOSC5 protein or human neuroblastoma SHEP cells expressing Dox-inducible MYCN wild-type (MYCNwt) or RNA-binding deficient triple mutant MYCN (MYCN3A), were grown in RPMI-1640 medium supplemented with with 10% bovine serum and 1% penicillin/ streptomycin. Mouse NIH3T3 spike-in cells were grown in DMEM medium supplemented with 10% bovine serum and 1% penicillin/ streptomycin. Cells were cultured at 37°C at 5% CO2.
Human neuroblastoma IMR-32 cells expressing doxycycline (Dox)-inducible shRNAs or expressing an endogenous AID-tagged EXOSC5 protein or human neuroblastoma SHEP cells expressing Dox-inducible MYCN wild-type (MYCNwt) or RNA-binding deficient triple mutant MYCN (MYCN3A), were grown in RPMI-1640 medium supplemented with with 10% bovine serum and 1% penicillin/ streptomycin. Mouse NIH3T3 spike-in cells were grown in DMEM medium supplemented with 10% bovine serum and 1% penicillin/ streptomycin. Cells were cultured at 37°C at 5% CO2.

extract_protocol:
For eCLIPseq, cells were UV-crosslinked at 254 nm, lysed, sonicated in a Covaris Ultrasonicator at low settings and mixed with pre-incubated primary antibody/beads. Size-matched input samples were transfered to new tubes, IP samples were dephosphorylated and 3'RNA adapter ligated. IP and input samples were mixed with LDS buffer, loaded onto Bis-Tris gels and transfered to nitrocellulose membranes. Membrane sections were incubated with Proteinase K, input samples were dephosphorylated and 3'RNA adapter ligated. All samples were then reverse transcribed and random 5' adapters were added.
For CUT&RUN (CnR), cells were coupled to ConA-coated magentic beads (Polysciences Europe), permeabilized and incubated with the primary antibody in antibody binding buffer. Lysates were washed in the presence of digitonin and MNase, then an incubation buffer was added and MNase activity was stopped by RNase A. Decrosslinking was done with SDS and Proteinase K and DNA was extracted with phenol-chloroform.
For ChIP-Rx, cells were fixed with formaldehyde and spiked with mouse NIH3T3 cellsat a 1:10 cell ratio. The crosslinked chromatin was extracted and fragmented using a Covaris Ultrasonicator. Chromatin was then added to a pre-incubated bead/antibody mix. Following incubation and stringent washing, chromatin was eluted, protein and RNA were digested with proteinase K and RNase A and DNA was isolated by phenol-chloroform.
For RNAseq, total RNA was isolated, purified and polyA-RNA was extracted using the NEBNext Poly(A) mRNA Magnetic Isolation Module. For 4sU-RNAseq and 3'RNAseq, cells were treated with 4sU and lysed either directly (3'RNAseq) or after a 120 min wash-out (4sU-RNAseq). Total RNA was isolated, biotinylated and isolated using streptavidin-conjugated magnetic beads. For 3'RNAseq, polyA tails were added using E.coli PolyA Polymerase, where indicated (PA).
ChIP-Rx and CnR libraries were prepared with the NEBNext Ultra II DNA Library Prep kit. 4sU-RNAseq and 3'RNAseq libraries were depleted of rRNA using the NEBNext rRNA Depletion Kit (Human/Mouse/Rat). RNAseq and 4sU-RNAseq libraries and were generated with the NEBNext Ultra II Directional RNA Library Prep kit,. 3'RNAseq libraries were prepared with the QuantSeq 3′ mRNA-Seq Library Prep Kit REV. For eCLIPseq, cDNA samples were amplified using Illumina compatible index primers.
For all described libraries, a qPCR was performed to determine optimal PCR cycle number. For eCLIP, cDNA samples were amplified and gel purified. All other libraries were bead purified. Library quality and fragment size distribution was analyzed on a Fragment Analyzer (Advanced Analytical). Sequencing was performed in paired-end (except for 3'RNAseq, which was done in single-read) mode on a NextSeq2000 platform (Illumina).

treatment_protocol:
The expression of shRNAs (shEXOSC10 or shMYCN) or MYCN constructs (wt or 3A) was induced with 1 µg/ml doxycycline. EXOSC5 AID was induced by 100 µM Indole-3-acetic acid (IAA).
The expression of shRNAs (shEXOSC10 or shMYCN) or MYCN constructs (wt or 3A) was induced with 1 µg/ml doxycycline. EXOSC5 AID was induced by 100 µM Indole-3-acetic acid (IAA).

value: SHEP

value: Neuroblastoma

tag: iaa treatment_(exosc5_aid_active)

value: No

tag: dox treatment_(shrbm7_active)

tag: dox treatment_(mycnwt_active)

tag: dox treatment_(mycn3a_active)

tag: antibody


title: ConL8

tag: tissue


title: InfL19


title: RNAseq_noMYCN_R2

description: polyA-RNAseq

tag: cell type


title: RNAseq_noMYCN_R1

description: polyA-RNAseq


title: RNAseq_MYCNWT_R2

description: polyA-RNAseq

value: Yes


title: RNAseq_MYCNWT_R3

description: polyA-RNAseq


title: RNAseq_MYCNWT_R1

description: polyA-RNAseq


title: RNAseq_MYCN3A_R3

description: polyA-RNAseq


title: RNAseq_MYCN3A_R1

description: polyA-RNAseq


title: ConE46


title: CnR_SIN3A_EXOSC5AID_R1

description: Cut&Run

value: SIN3A (Novus Biologicals; NB600-1263)


title: eCLIP_MYCN_shRBM7_R1

description: eCLIPseq

value: MYCN (Santa Cruz Biotechnology; sc-53993)


title: eCLIP_MYCN_shRBM7_Input

description: eCLIPseq


title: eCLIP_MYCN_shRBM7_R2

description: eCLIPseq


title: CnR_SIN3A_Control_DMSO_R1

description: Cut&Run


title: CnR_SIN3A_EXOSC5AID_R2

description: Cut&Run


title: 1ng_MeOH_HEK293T_Buffer_only_replicate3

data_processing:
remove adapter with Cutadapt
align to hg38 using STAR 2.4.0 (Homo sapiens)
SAILOR analysis to call C-to-U edits and keep only sites with score >0.5 and edit fraction <80%
FLARE analysis to call C-to-U edit clusters
Intersect the edit clusters from 3 replicates, which yields "*confident_peaks.bed"
Subtract STAMP confident clusters to Buffer only control, which yields "*cleaned_confident_peaks.bed"
Assembly: hg38
Supplementary files format and content: SAILOR step yields bed file: *0.5Score0.8Fraction.fastqTr.sorted.STARUnmapped.out.sorted.STARAligned.out.sorted.bam.combined.readfiltered.formatted.varfiltered.snpfiltered.ranked.bed
Supplementary files format and content: FLARE step yields .tsv file: "*merged_sorted_peaks.fdr_0.1.d_15.scored.tsv"
Supplementary files format and content: Intersection step yields .bed file:  "*confident_peaks.bed"
Supplementary files format and content: Subtraction step yields .bed file:  "*cleaned_confident_peaks.bed"

extract_protocol:
Direct-zol RNA Miniprep Kits, Zymo Research (Cat # R2052)
NEBNext® Single Cell/Low Input RNA Library Prep Kit (NEB)


title: CnR_IgG_SIN3A_EXOSC5AID_R2

description: Cut&Run

value: Pooled

value: Murine IgG Control Antibody (Sigma-Aldrich; I5381)


title: 1ng_MeOH_HEK293T_RBFOX2_replicate3

description:
1ng_RBFOX2_confident_peaks.bed
1ng_RBFOX2_cleaned_confident_peaks.bed


title: CnR_IgG_SIN3A_EXOSC5AID_R1

description: Cut&Run


title: 1ng_MeOH_HEK293T_Buffer_only_replicate2


title: 100pg_MeOH_HEK293T_Buffer_only_replicate3


title: 100pg_MeOH_HEK293T_Buffer_only_replicate2


title: 1ng_MeOH_HEK293T_Enzyme_only


title: 1ng_MeOH_HEK293T_RBFOX2_replicate1


title: 100pg_MeOH_HEK293T_Buffer_only_replicate1


title: 100pg_MeOH_HEK293T_RBFOX2_replicate1

description:
100pg_RBFOX2_confident_peaks.bed
100pg_RBFOX2_cleaned_confident_peaks.bed


title: 100pg_MeOH_HEK293T_RBFOX2_replicate2


title: eCLIP_MYCN_shZCCHC8_R2

description: eCLIPseq

data_processing:
Fastq files were generated Illumina’s BaseSpace platform and overall sequencing quality was analyzed with the FastQC script.
For RNA libraries from the eCLIP experiment, reads were aligned to hg19 and mm10 using STAR 2.7.6a. Dataset was normalized based on mm10 reads.
Following normalization, alignment files were converted to bigwig with deeptools bamCoverage v3.5.1. CLIPper v2.1.0 was used for eCLIP-seq peak calling.
Assembly: hg19, mm10
Supplementary files format and content: Files are in bigwig format.

growth_protocol: Human neuroblastoma IMR-32 cells expressing doxycycline (Dox)-inducible shRNAs were grown in RPMI-1640 medium supplemented with with 10% bovine serum and 1% penicillin/ streptomycin. Cells were cultured at 37°C at 5% CO2.

extract_protocol:
For eCLIPseq, cells were UV-crosslinked at 254 nm, lysed, sonicated in a Covaris Ultrasonicator at low settings and mixed with pre-incubated primary antibody/beads. Size-matched input samples were transfered to new tubes, IP samples were dephosphorylated and 3'RNA adapter ligated. IP and input samples were mixed with LDS buffer, loaded onto Bis-Tris gels and transfered to nitrocellulose membranes. Membrane sections were incubated with Proteinase K, input samples were dephosphorylated and 3'RNA adapter ligated. All samples were then reverse transcribed and random 5' adapters were added.
A qPCR was performed to determine optimal PCR cycle number. For eCLIP, cDNA samples were amplified and gel purified. Library quality and fragment size distribution was analyzed on a Fragment Analyzer (Advanced Analytical). Sequencing was performed in paired-end mode on a NextSeq2000 platform (Illumina).

treatment_protocol: The expression of shRNAs (shZCCHC8) was induced with 1 µg/ml doxycycline.

tag: dox treatment_(shzcchc8_active)

value: Dox

tag: dox treatment_(shmycn_active)

value: not aplicable

tag: ha-exosc10 mutant_expressed

tag: polya-polymerase used


title: eCLIP_MYCN_shZCCHC8_Input

description: eCLIPseq

tag: cell line


title: 3'RNAseq_shEXOSC10_noPA_R2

data_processing:
Fastq files were generated Illumina’s BaseSpace platform and overall sequencing quality was analyzed with the FastQC script.
For CnR and ChIP-Rx, reads were aligned to hg19 and mm10 using Bowtie2 v2.3.5.1 with default parameters. For RNA, 4sU and 3'RNAseq reads were aligned to hg19 and mm10 using STAR v2.7.10a (2.7.6a for eCLIP). For 4sUseq reads mapping to rRNA, exons and blacklist regions were removed. All datasets (except the SIN3A and MNT CnR, which were normalized to sequencing depth) were normalized based on mm10 reads.
Following normalization, alignment files were converted to bigwig with deeptools bamCoverage v3.5.1. 3'RNAseq stranded bedgraphs were generated with a custom python script and bedgraphs were then converted to bigwig with bedGraphToBigWig v2.9. CLIPper v2.1.0 was used for eCLIP-seq peak calling. featureCounts v2.0.3 was used for RNAseq counts.
Assembly: hg19, mm10
Supplementary files format and content: For ChIP-RX, eCLIP, 4sU-RNA and 3'RNA processed files are in bigwig format. All CnR processed files are also in bigwig format, except for SIN3A, MNT and MAX R2 files, which are in bedgraph format. The RNAseq processed file is a featureCounts output text file.

growth_protocol: Human neuroblastoma IMR-32 cells expressing doxycycline (Dox)-inducible shRNAs or expressing an endogenous AID-tagged EXOSC5 protein, were grown in RPMI-1640 medium supplemented with with 10% bovine serum and 1% penicillin/ streptomycin. Mouse NIH3T3 spike-in cells were grown in DMEM medium supplemented with 10% bovine serum and 1% penicillin/ streptomycin. Cells were cultured at 37°C at 5% CO2.

extract_protocol:
For eCLIPseq, cells were UV-crosslinked at 254 nm, lysed, sonicated in a Covaris Ultrasonicator at low settings and mixed with pre-incubated primary antibody/beads. Size-matched input samples were transfered to new tubes, IP samples were dephosphorylated and 3'RNA adapter ligated. IP and input samples were mixed with LDS buffer, loaded onto Bis-Tris gels and transfered to nitrocellulose membranes. Membrane sections were incubated with Proteinase K, input samples were dephosphorylated and 3'RNA adapter ligated. All samples were then reverse transcribed and random 5' adapters were added.
For Cut&Run (CnR), cells were coupled to ConA-coated magentic beads (Polysciences Europe), permeabilized and incubated with the primary antibody in antibody binding buffer. Lysates were washed in the presence of digitonin and MNase, then an incubation buffer was added and MNase activity was stopped by RNase A. Decrosslinking was done with SDS and Proteinase K and DNA was extracted with phenol-chloroform.
For ChIP-Rx, cells were fixed with formaldehyde and spiked with mouse NIH3T3 cellsat a 1:10 cell ratio. The crosslinked chromatin was extracted and fragmented using a Covaris Ultrasonicator. Chromatin was then added to a pre-incubated bead/antibody mix. Following incubation and stringent washing, chromatin was eluted, protein and RNA were digested with proteinase K and RNase A and DNA was isolated by phenol-chloroform.
For RNAseq, total RNA was isolated, purified and polyA-RNA was extracted using the NEBNext Poly(A) mRNA Magnetic Isolation Module. For 4sU-RNAseq and 3'RNAseq, cells were treated with 4sU and lysed either directly (3'RNAseq) or after a 120 min wash-out (4sU-RNAseq). Total RNA was isolated, biotinylated and isolated using streptavidin-conjugated magnetic beads. For 3'RNAseq, polyA tails were added using E.coli PolyA Polymerase, where indicated (PA).
ChIP-Rx and CnR libraries were prepared with the NEBNext Ultra II DNA Library Prep kit. 4sU-RNAseq and 3'RNAseq libraries were depleted of rRNA using the NEBNext rRNA Depletion Kit (Human/Mouse/Rat). RNAseq and 4sU-RNAseq libraries and were generated with the NEBNext Ultra II Directional RNA Library Prep kit,. 3'RNAseq libraries were prepared with the QuantSeq 3′ mRNA-Seq Library Prep Kit REV. For eCLIPseq, cDNA samples were amplified using Illumina compatible index primers.
For all described libraries, a qPCR was performed to determine optimal PCR cycle number. For eCLIP, cDNA samples were amplified and gel purified. All other libraries were bead purified. Library quality and fragment size distribution was analyzed on a Fragment Analyzer (Advanced Analytical). Sequencing was performed in paired-end (except for 3'RNAseq, which was done in single-read) mode on a NextSeq2000 platform (Illumina).

treatment_protocol: The expression of shRNAs (shEXOSC10 or shMYCN) was induced with 1 µg/ml doxycycline. EXOSC5 AID was induced by 100 µM Indole-3-acetic acid (IAA).

tag: dox treatment_(shexosc10_active)

tag: fraction

value: 3' RNA

tag: assay

value: 3' RNAseq


title: eCLIP_MYCN_shZCCHC8_R1

description: eCLIPseq


title: 3'RNAseq_shEXOSC10_noPA_R1


title: RNAseq_MYCN3A_R2

description: polyA-RNAseq


title: 1ng_MeOH_HEK293T_Buffer_only_replicate1

tag: replicate


title: CnR_SIN3A_Control_DMSO_R2

description: Cut&Run


title: 1ng_MeOH_HEK293T_RBFOX2_replicate2


title: 3'RNAseq_shMYCN_noPA_R1


title: 3'RNAseq_shEXOSC10_PA_R2


title: 3'RNAseq_Control_noPA_R1


title: 3'RNAseq_shMYCN_noPA_R2


title: 3'RNAseq_shMYCN_PA_R1


title: 3'RNAseq_Control_PA_R2


title: 3'RNAseq_Control_noPA_R2


title: 3'RNAseq_shEXOSC10_PA_R1


title: 3'RNAseq_Control_PA_R1


title: 100pg_MeOH_HEK293T_RBFOX2_replicate3


title: eCLIP_EXOSC10mut_shMYCN_R2

value: protein-bound RNA

value: eCLIPseq


title: eCLIP_EXOSC10mut_Control_R2


title: eCLIP_EXOSC10mut_Control_R1


title: eCLIP_EXOSC10mut_shMYCN_Input


title: eCLIP_EXOSC10mut_shMYCN_R1


title: eCLIP_EXOSC10mut_Control_Input


title: 100pg_MeOH_HEK293T_Enzyme_only


title: RNAseq_shEXOSC10_R1

value: polyA RNA

value: polyA-RNAseq


title: Donor 2, B cells, BTKi, anti-BCR

description: Donor 2 aBCR BTKi

tag: individual

value: Donor 2

value: Blood

value: B cells

tag: disease state

value: Healthy

tag: treatment

value: Anti-BCR BTKi

tag: time


title: RNAseq_Control_R3

value: Neuroblastoma

value: No

tag: iaa treatment_(exosc5_aid_active)


title: RNAseq_Control_R2

tag: antibody


title: CnR_MAX_shEXOSC10_R2

data_processing:
Fastq files were generated Illumina’s BaseSpace platform and overall sequencing quality was analyzed with the FastQC script.
For CnR and ChIP-Rx, reads were aligned to hg19 and mm10 using Bowtie2 v2.3.5.1 with default parameters. For RNA, 4sU and 3'RNAseq reads were aligned to hg19 and mm10 using STAR v2.7.10a (2.7.6a for eCLIP). For 4sUseq reads mapping to rRNA, exons and blacklist regions were removed. All datasets (except the SIN3A and MNT CnR, which were normalized to sequencing depth) were normalized based on mm10 reads.
Following normalization, alignment files were converted to bigwig with deeptools bamCoverage v3.5.1. 3'RNAseq stranded bedgraphs were generated with a custom python script and bedgraphs were then converted to bigwig with bedGraphToBigWig v2.9. CLIPper v2.1.0 was used for eCLIP-seq peak calling. featureCounts v2.0.3 was used for RNAseq counts.
Assembly: hg19, mm10
Supplementary files format and content: For ChIP-RX, eCLIP, 4sU-RNA and 3'RNA processed files are in bigwig format. All CnR processed files are also in bigwig format, except for SIN3A, MNT and MAX R2 files, which are in bedgraph format. The RNAseq processed file is a featureCounts output text file.
Library strategy: Cut&Run

value: MAX (Proteintech; 10426-1-AP)

value: Cut&Run


title: CnR_MAX_Control_R2


title: 3'RNAseq_shMYCN_PA_R2

value: Dox

value: Yes


title: CnR_MNT_Control_R2

value: MNT (Thermo Fisher Scientific; A303-627A-T)


title: RNAseq_Control_R1


title: CnR_SIN3A_Control_R2

value: SIN3A (Novus Biologicals; NB600-1263)


title: CnR_MNT_shEXOSC10_R2


title: CnR_SIN3A_shEXOSC10_R2


title: CnR_SIN3A_shEXOSC10_R1


title: CnR_MNT_Control_R1


title: CnR_MNT_shEXOSC10_R1


title: CnR_IgG_R2

value: Pooled

value: Murine IgG Control Antibody (Sigma-Aldrich; I5381)


title: CnR_IgG_R3


title: CnR_IgG_R1


title: ChIPRx_MYCN_shEXOSC10_R2

value: MYCN (Santa Cruz Biotechnology; sc-53993)

value: ChIP-Rx


title: RNAseq_shEXOSC10_R3

value: polyA RNA

value: polyA-RNAseq


title: Donor 3, B cells, Vehicle, anti-BCR

description: Donor 3 aBCR Vehicle

value: Donor 3

tag: tissue

value: Blood

value: B cells

value: Anti-BCR Vehicle


title: CnR_MAX_Control_R1

tag: cell type

value: Neuroblastoma

value: No


title: RNAseq_shEXOSC10_R2


title: ChIPRx_MYCN_shEXOSC10_R1


title: ChIPRx_MYCN_Input_R2


title: ChIPRx_MYCN_Control_R1


title: ChIPRx_MYCN_Control_R2


title: eCLIP_IgG_R2

value: protein-bound RNA

value: eCLIPseq


title: eCLIP_IgG_R1


title: ChIPRx_MYCN_Input_R1


title: eCLIP_U2OS_Control_R2


title: eCLIP_U2OS_Control_R1


title: CnR_SIN3A_Control_R1

value: Cut&Run


title: eCLIP_MYCN_shEXOSC10_Input


title: eCLIP_MYCN_shEXOSC10_R1

value: Dox


title: eCLIP_U2OS_Control_Input


title: CnR_MAX_shEXOSC10_R1

value: MAX (Proteintech; 10426-1-AP)


title: ChIPRx_MYCN_Control_R3


title: eCLIP_MYCN_EXOSC5AID_R1

value: IAA


title: eCLIP_MYCN_EXOSC5AID_R2


title: ChIPRx_MYCN_shEXOSC10_R3


title: eCLIP_MYCN_Control_R1


title: 4sUseq_shEXOSC10_R3

value: nascent RNA


title: eCLIP_MYCN_EXOSC5AID_Input

value: Pooled

value: MYCN (Santa Cruz Biotechnology; sc-53993)


title: 4sUseq_shEXOSC10_R2


title: 4sUseq_Control_R2


title: 4sUseq_Control_R3


title: 4sUseq_shEXOSC10_R1


title: eCLIP_MYCN_Control_Input


title: HEK293T_buffer_only_INSCRIBE_rep1

description: Long-read HEK293T buffer only INSCRIBE rep1

data_processing:
CCS calling (v8.0.0)
read deconcatenation using skera(1.2.0)
read trimming with lima(v2.9.0)
refine reads with isoseq refine (v4.1.1)
read alignment with pbmm2 (v1.13.0)
QC with NanoPlot (v1.32.1)
filter unmapped reads, supplementary alignment reads, reads aligned to the incorrect strand, and reads having read quality below 20
call edits using custom script
remove SNPs
Assembly: hg38
Supplementary files format and content: isoforms level edit detection (.bed)

extract_protocol:
Direct-zol RNA Miniprep Kits, Zymo Research (Cat # R2052)
Kinnex full-length RNA kit (PN 103-072-000)

value: Kidney

value: immortalized cell line

tag: molecule subtype

value: rRNA depleted RNA


title: HEK293T_APO_only_INSCRIBE_rep3

description: Long-read HEK293T APO only INSCRIBE rep3


title: HEK293T_buffer_only_INSCRIBE_rep2

description: Long-read HEK293T buffer only INSCRIBE rep2


title: HEK293T_APO_only_INSCRIBE_rep2

description: Long-read HEK293T APO only INSCRIBE rep2


title: 4sUseq_Control_R1

tag: dox treatment_(shmycn_active)


title: eCLIP_IgG_Input

tag: ha-exosc10 mutant_expressed

value: Murine IgG Control Antibody (Sigma-Aldrich; I5381)


title: HEK293T_RBFOX2_INSCRIBE_rep2

description: Long-read HEK293T RBFOX2 INSCRIBE rep2


title: eCLIP_MYCN_Control_R2

tag: polya-polymerase used


title: eCLIP_MYCN_shEXOSC10_R2

tag: cell line


title: Blood,excercised,cell869,wk12

city: Sydney

first: Rowena

last: Bull

state: NSW

address: Wallace Wurth Building (C27)

country: Australia

institute: University of New South Wales

description: SMARTseq2 single cell RNA sequencing data from E2-specific memory B cells

data_processing:
The raw data was aligned with human genome to map the heavy and light chain genes which were further annotated using IgBlast software implemented in VDJPuzzle
Inhouse pipeline used to generate the matrix that was further processed using seurat and other packages for paired-end sequences
Assembly: Sequences were processed using VDJPuzzle for BCR data and inhouse pipelines for Gene expression analysis
Supplementary files format and content: barcodes.tsv
Supplementary files format and content: features.tsv
Supplementary files format and content: matrix.mtx

extract_protocol:
Seven to ten million cryopreserved PBMCs were sorted for E2-specific B cells as described previously. S figure 4, in the article indicates the time points selected for flow analysis and transcriptomics/repertoire analysis. In brief, PBMCs were stained with an in-house developed rE2 cocktail tetramer (Gt1a [H77] and Gt3a [UKN3A13.6]) as well as anti-CD19, anti-CD20, anti-CD10, anti-CD38, anti-IgD, anti-IgG, anti-CD27,anti-CD21 and anti-CD95 antibodies, and single cell sorted using flow cytometry. Population phenotypes were analysed with FlowJo version 10 software (Tree Star Inc.) (See CTAT Table for antibody details and see S Figure 3 for gating strategy and how gates were set for CD95 positivity based on FMO).
E2+ B cells were sorted into single wells and single cell RNA sequencing was performed following the smartseq2 protocol as previously described. BCR sequences were reconstructed using VDJpuzzle 2.0. Identification of B cell clonotypes and SHM across infections was done using Immcantation framework.  Paired-end reads from Smart-seq2 were aligned with STAR (version 2.7.1a) using the GRCh38.p13 human reference genome. Transcripts per Million (TPM) were quantified using RSEM (version 1.2.28) using rsem-calculate-expression command on HPC clusters.

value: Blood

value: human samples

value: Memory B cells

tag: genotype


title: Blood,excercised,cell867,wk12


title: Blood,excercised,cell868,wk12


title: Blood,excercised,cell865,wk12


title: Blood,excercised,cell870,wk12


title: Blood,excercised,cell864,wk12


title: Blood,excercised,cell860,wk12


title: Blood,excercised,cell861,wk12


title: Blood,excercised,cell862,wk12


title: Blood,excercised,cell859,wk12


title: Blood,excercised,cell866,wk12


title: Blood,excercised,cell863,wk12


title: Blood,excercised,cell858,wk12


title: Blood,excercised,cell855,wk12


title: Blood,excercised,cell856,wk12


title: Blood,excercised,cell854,wk12


title: Blood,excercised,cell857,wk12


title: Blood,excercised,cell850,wk12


title: HEK293T_RBFOX2_INSCRIBE_rep1

description: Long-read HEK293T RBFOX2 INSCRIBE rep1


title: Blood,excercised,cell852,wk12


title: Blood,excercised,cell851,wk12


title: Blood,excercised,cell845,wk12


title: Blood,excercised,cell847,wk12


title: Blood,excercised,cell844,wk12


title: Blood,excercised,cell846,wk12


title: Blood,excercised,cell849,wk12


title: Blood,excercised,cell843,wk12


title: Blood,excercised,cell839,wk12


title: Blood,excercised,cell842,wk12


title: Blood,excercised,cell841,wk12


title: Blood,excercised,cell836,wk12


title: Blood,excercised,cell840,wk12


title: Blood,excercised,cell837,wk12


title: Blood,excercised,cell838,wk12


title: Blood,excercised,cell833,wk12


title: Blood,excercised,cell835,wk12


title: Blood,excercised,cell832,wk12


title: Blood,excercised,cell853,wk12


title: Blood,excercised,cell834,wk12


title: Blood,excercised,cell830,wk12


title: Blood,excercised,cell828,wk12


title: Blood,excercised,cell848,wk12


title: Blood,excercised,cell831,wk12


title: Blood,excercised,cell825,wk12


title: Blood,excercised,cell826,wk12


title: Blood,excercised,cell829,wk12


title: Blood,excercised,cell823,wk12


title: Blood,excercised,cell824,wk12


title: Blood,excercised,cell822,wk12


title: Blood,excercised,cell819,wk12


title: Blood,excercised,cell818,wk12


title: Blood,excercised,cell817,wk12


title: Blood,excercised,cell820,wk12


title: Blood,excercised,cell816,wk12


title: Blood,excercised,cell815,wk12


title: Blood,excercised,cell814,wk12


title: Blood,excercised,cell813,wk12


title: Blood,excercised,cell812,wk12


title: Blood,excercised,cell811,wk12


title: Blood,excercised,cell810,wk12


title: Blood,excercised,cell808,wk12


title: Blood,excercised,cell807,wk12


title: Blood,excercised,cell806,wk12


title: Blood,excercised,cell827,wk12


title: Blood,excercised,cell805,wk12


title: Blood,excercised,cell804,wk12


title: Blood,excercised,cell803,wk12


title: Blood,excercised,cell801,wk12


title: Blood,excercised,cell821,wk12


title: Blood,excercised,cell798,wk12


title: Blood,excercised,cell802,wk12


title: Blood,excercised,cell799,wk12


title: Blood,excercised,cell800,wk12


title: Blood,excercised,cell795,wk12


title: Blood,excercised,cell796,wk12


title: Blood,excercised,cell790,wk02nd_infec


title: Blood,excercised,cell788,wk02nd_infec


title: Blood,excercised,cell791,wk02nd_infec


title: Blood,excercised,cell794,wk12


title: Blood,excercised,cell797,wk12


title: Blood,excercised,cell792,wk02nd_infec


title: Blood,excercised,cell793,wk12


title: Blood,excercised,cell786,wk02nd_infec


title: Blood,excercised,cell789,wk02nd_infec


title: Blood,excercised,cell784,wk02nd_infec


title: Blood,excercised,cell787,wk02nd_infec


title: Blood,excercised,cell809,wk12


title: Blood,excercised,cell785,wk02nd_infec


title: Blood,excercised,cell780,wk02nd_infec


title: Blood,excercised,cell782,wk02nd_infec


title: Blood,excercised,cell783,wk02nd_infec


title: Blood,excercised,cell779,wk02nd_infec


title: Blood,excercised,cell778,wk02nd_infec


title: Blood,excercised,cell777,wk02nd_infec


title: Blood,excercised,cell771,wk02nd_infec


title: Blood,excercised,cell774,wk02nd_infec


title: Blood,excercised,cell769,wk02nd_infec


title: Blood,excercised,cell770,wk02nd_infec


title: Blood,excercised,cell776,wk02nd_infec


title: Blood,excercised,cell773,wk02nd_infec


title: Blood,excercised,cell766,wk02nd_infec


title: Blood,excercised,cell764,wk02nd_infec


title: Blood,excercised,cell768,wk02nd_infec


title: Blood,excercised,cell763,wk02nd_infec


title: Blood,excercised,cell762,wk02nd_infec


title: Blood,excercised,cell761,wk02nd_infec


title: Blood,excercised,cell760,wk02nd_infec


title: Blood,excercised,cell781,wk02nd_infec


title: Blood,excercised,cell756,wk02nd_infec


title: Blood,excercised,cell757,wk02nd_infec


title: Blood,excercised,cell759,wk02nd_infec


title: Blood,excercised,cell758,wk02nd_infec


title: Blood,excercised,cell755,wk02nd_infec


title: Blood,excercised,cell775,wk02nd_infec


title: Blood,excercised,cell754,wk02nd_infec


title: Blood,excercised,cell772,wk02nd_infec


title: Blood,excercised,cell751,wk02nd_infec


title: Blood,excercised,cell753,wk02nd_infec


title: Blood,excercised,cell752,wk02nd_infec


title: Blood,excercised,cell750,wk02nd_infec


title: Blood,excercised,cell767,wk02nd_infec


title: Blood,excercised,cell748,wk02nd_infec


title: Blood,excercised,cell749,wk02nd_infec


title: Blood,excercised,cell744,wk02nd_infec


title: Blood,excercised,cell743,wk02nd_infec


title: Blood,excercised,cell765,wk02nd_infec


title: Blood,excercised,cell747,wk02nd_infec


title: Blood,excercised,cell745,wk02nd_infec


title: Blood,excercised,cell741,wk02nd_infec


title: Blood,excercised,cell742,wk02nd_infec


title: Blood,excercised,cell738,wk02nd_infec


title: Blood,excercised,cell740,wk02nd_infec


title: Blood,excercised,cell739,wk02nd_infec


title: Blood,excercised,cell737,wk02nd_infec


title: Blood,excercised,cell736,wk02nd_infec


title: Blood,excercised,cell734,wk02nd_infec


title: Blood,excercised,cell735,wk02nd_infec


title: Blood,excercised,cell733,wk02nd_infec


title: Blood,excercised,cell730,wk02nd_infec


title: Blood,excercised,cell731,wk02nd_infec


title: Blood,excercised,cell732,wk02nd_infec


title: Blood,excercised,cell728,wk02nd_infec


title: Blood,excercised,cell727,wk02nd_infec


title: Blood,excercised,cell724,wk4


title: Blood,excercised,cell729,wk02nd_infec


title: Blood,excercised,cell726,wk02nd_infec


title: Blood,excercised,cell725,wk02nd_infec


title: Blood,excercised,cell746,wk02nd_infec


title: Blood,excercised,cell721,WK24rpt


title: Blood,excercised,cell719,WK24rpt


title: Blood,excercised,cell720,WK24rpt


title: Blood,excercised,cell716,WK24rpt


title: Blood,excercised,cell715,WK24rpt


title: Blood,excercised,cell718,WK24rpt


title: Blood,excercised,cell714,WK24rpt


title: Blood,excercised,cell708,WK24rpt


title: Blood,excercised,cell712,WK24rpt


title: Blood,excercised,cell723,WK24rpt


title: Blood,excercised,cell710,WK24rpt


title: Blood,excercised,cell707,WK24rpt


title: Blood,excercised,cell717,WK24rpt


title: Blood,excercised,cell709,WK24rpt


title: Blood,excercised,cell713,WK24rpt


title: Blood,excercised,cell706,WK24rpt


title: Blood,excercised,cell705,WK24rpt


title: Blood,excercised,cell703,WK24rpt


title: Blood,excercised,cell699,WK24rpt


title: Blood,excercised,cell700,WK24rpt


title: Blood,excercised,cell704,WK24rpt


title: Blood,excercised,cell702,WK24rpt


title: Blood,excercised,cell722,WK24rpt


title: Blood,excercised,cell696,WK24rpt


title: Blood,excercised,cell697,WK24rpt


title: Blood,excercised,cell692,WK24rpt


title: Blood,excercised,cell693,WK24rpt


title: Blood,excercised,cell698,WK24rpt


title: Blood,excercised,cell691,WK24rpt


title: Blood,excercised,cell701,WK24rpt


title: Blood,excercised,cell695,WK24rpt


title: Blood,excercised,cell690,WK24rpt


title: Blood,excercised,cell694,WK24rpt


title: Blood,excercised,cell688,WK24rpt


title: Blood,excercised,cell689,WK24rpt


title: Blood,excercised,cell711,WK24rpt


title: Blood,excercised,cell686,WK24rpt


title: Blood,excercised,cell687,WK24rpt


title: Blood,excercised,cell681,WK12


title: Blood,excercised,cell685,WK24rpt


title: Blood,excercised,cell680,WK12


title: Blood,excercised,cell684,WK24rpt


title: Blood,excercised,cell679,WK12


title: Blood,excercised,cell678,WK12


title: Blood,excercised,cell677,WK12


title: Blood,excercised,cell676,WK12


title: Blood,excercised,cell674,WK12


title: Blood,excercised,cell673,WK12


title: Blood,excercised,cell675,WK12


title: Blood,excercised,cell672,WK12


title: Blood,excercised,cell671,WK12


title: Blood,excercised,cell669,WK12


title: Blood,excercised,cell667,WK2


title: Blood,excercised,cell670,WK12


title: Blood,excercised,cell665,WK2


title: Blood,excercised,cell666,WK2


title: Blood,excercised,cell668,WK2


title: Blood,excercised,cell663,WK2


title: Blood,excercised,cell661,WK2


title: Blood,excercised,cell682,WK12


title: Blood,excercised,cell683,WK12


title: Blood,excercised,cell664,WK2


title: Blood,excercised,cell659,WK2


title: Blood,excercised,cell658,WK2


title: Blood,excercised,cell657,WK2


title: Blood,excercised,cell660,WK2


title: Blood,excercised,cell662,WK2


title: Blood,excercised,cell655,WK2


title: Blood,excercised,cell654,WK2


title: Blood,excercised,cell651,WK2


title: Blood,excercised,cell656,WK2


title: Blood,excercised,cell652,WK2


title: Blood,excercised,cell650,WK2


title: Blood,excercised,cell649,WK2


title: Blood,excercised,cell653,WK2


title: Blood,excercised,cell648,WK2


title: Blood,excercised,cell646,WK2


title: Blood,excercised,cell645,WK2


title: Blood,excercised,cell644,WK2


title: Blood,excercised,cell642,WK2


title: Blood,excercised,cell647,WK2


title: Blood,excercised,cell641,WK2


title: Blood,excercised,cell638,WK2


title: Blood,excercised,cell640,WK2


title: Blood,excercised,cell639,WK2


title: Blood,excercised,cell643,WK2


title: Blood,excercised,cell637,WK2


title: Blood,excercised,cell635,WK2


title: Blood,excercised,cell631,WK2


title: Blood,excercised,cell627,24M1


title: Blood,excercised,cell634,WK2


title: Blood,excercised,cell633,WK2


title: Blood,excercised,cell625,24M1


title: Blood,excercised,cell630,WK2


title: Blood,excercised,cell626,24M1


title: Blood,excercised,cell624,24M1


title: Blood,excercised,cell623,24M1


title: Bulk BCR, adult male, subject P11, PBMC, year 2 day-6

city: New Haven

first: Steven

last: Kleinstein

state: Ct

address: 300 George St.

department: Pathology

institute: Yale University

description:
P11_BCR.tsv
P11_PBMC-ASC_d6_Y2

data_processing:
Bulk BCR: Demultiplexed pair-end reads were preprocessed using pRESTO v.0.5.7. Initial germline V(D)J gene annotation was  performed using IgBLAST v1.9.0. IgBLAST output was processed using  Change-O v0.4.0.
scBCR: Demultiplexed pair-end FASTQ reads were preprocessed using the “cellranger vdj” command from 10 × Genomics’ Cell Ranger v.3.1.0 for alignment against the GRCh38 human reference v.3.1.0 (refdata-cellranger-vdj-GRCh38-alts-ensembl-3.1.0). Initial germline V(D)J gene annotation was performed as described for bulk sequencing BCR reads
Processed BCR sequences from bulk sequencing and single-cell sequencing (Extended Data Table 4), were combined with monoclonal antibody sequences for further analysis.
Combined BCR sequences were called for genotyping using the function findNovelAlleles() and inferGenotype() from TIgGER v.0.2.11 ([MH5] ). Individual genotypes, including novel V gene alleles missing from IMGT/ GENE-DB, were computationally inferred and used to finalize V(D)J annotations.
Assembly: GRCh38
Supplementary files format and content: tar.gz: compressed archive of 10X Genomics Cell Ranger output files.
Supplementary files format and content: tsv: AIRR-seq rearrangement format

source_name: PBMC

extract_protocol:
PBMCs were isolated from blood. Fine-needle aspiration was performed on axillary lymph nodes. RNA was purified from whole PBMCs and sorted PBs from participants using the RNeasy Micro Kit (Qiagen).
Bulk BCR: Reverse transcription, unique molecular identifier (UMI) barcoding, cDNA amplification, and Illumina linker addition to B cell heavy chain transcripts were performed using the human NEBNext Immune Sequencing Kit (NEB) according to the manufacturer’s instructions.  scBCR libraries were prepared as recommended by the 10× Genomics Chromium Single Cell V(D)J Reagent Kit (v1 Chemistry) user guide, with appropriate modifications to the PCR cycles based on the calculated cDNA concentration

value: PBMC

tag: Sex

value: male

tag: age

tag: isolate


title: Bulk BCR, adult male, subject P11, PBMC, year 1 day-0

description:
P11_BCR.tsv
P11_PBMC_d0_Y1


title: Blood,excercised,cell620,24M1


title: Blood,excercised,cell622,24M1


title: Blood,excercised,cell621,24M1


title: Blood,excercised,cell619,24M1


title: Bulk BCR, adult male, subject P11, PBMC, year 1 day-6

description:
P11_BCR.tsv
P11_PBMC-ASC_d6_Y1


title: Blood,excercised,cell618,24M1


title: Bulk BCR, adult male, subject P05, PBMC, year 1 day-0

description:
P05_BCR.tsv
P05_PBMC_d0_Y1


title: Blood,excercised,cell632,WK2


title: Blood,excercised,cell636,WK2


title: Blood,excercised,cell617,24M1


title: Bulk BCR, adult male, subject P05, PBMC, year 1 day-5

description:
P05_BCR.tsv
P05_PBMC-ASC_d5_Y1


title: Blood,excercised,cell629,WK2


title: Blood,excercised,cell628,24M1


title: Blood,excercised,cell615,24M1


title: Blood,excercised,cell616,24M1


title: Bulk BCR, adult male, subject P04, PBMC, year 2 day-7

description:
P04_BCR.tsv
P04_PBMC-ASC_d7_Y2


title: Bulk BCR, adult male, subject P04, PBMC, year 1 day-0

description:
P04_BCR.tsv
P04_PBMC_d0_Y1


title: Blood,excercised,cell613,24M1


title: Blood,excercised,cell612,24M1


title: Blood,excercised,cell614,24M1


title: ComXoxp_M17X

city: Louvain-la-Neuve

first: Frédéric

last: Toussaint

address: Place de l'Université 1

country: Belgium

institute: UCLouvain

data_processing:
Raw data were processed on the Galaxy server (use.galaxy.org) using Bowtie2 algorithm to yield BAM files
BAM files were analyzed using Seqmonk to count the number of reads per coding sequence
The dataset was exported into Excel for further analyses
Assembly: SL12653_transcripts_fasta.txt (DGCC12653)
Supplementary files format and content: the xls file (sheet 1) reporting RNAseq data is organized in columns as follows: column A: locus tag, column B: raw counts of WT grown in CDM*-DEB, column C: raw counts of WT grown in CDM*, column D: raw counts of WT (empty vector) grown in M17X, column E: raw counts of DcodY grown in CDM*-DEB, column F: raw counts of DcovRS grown in CDM*, column G: raw counts of WT overexpressing comX grown in M17X, column H: normalized counts of WT grown in CDM*-DEB, column I: normalized counts of WT grown in CDM*, column J: normalized counts of WT (empty vector) grown in M17X, column K: normalized counts of DcodY grown in CDM*-DEB, column L: normalized counts of DcovRS grown in CDM*, column M: normalized counts of WT overepressing comX grown in M17X, column N: ratio between normalized counts of DcodY and WT, column O: ratio between normalized counts of  DcovRS and WT, column P: ratio between normalized counts of comX overpression and WT.

organism: Lactococcus lactis

source_name: bacterial cell

growth_protocol: codY- and covRS-deletion mutants and wild-type controls were grown at 30°C without agitation in their respective efficient competence-activating medium (CDM*-DEB and CDM* for DcodY mutant and DcovRS mutant, respectively). The WT strain carrying an empty vector and the strain overexpressing the master comptence regulator ComX ((PxylT-comX fusion carried by a plasmid) were grown as reported above but in M17X medium containing Xylose as inducer.

extract_protocol:
RNA was harvested using Rneasy plus according to manufacturer indications. Samples were adjusted to 100 ng/µl and checked for integrity with an RNA Nano Chip (Agilent Technologies).
RNA sequencing library preparation was prepared using NEBNext rRNA Depletion Kit (Bacteria)  and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina following manufacturer’s instructions (NEB, Ipswich, MA, USA)
The sequencing libraries were multiplexed and clustered on the flowcell. After clustering, the flowcell was loaded on the Illumina NovaSeq 6000 instrument according to manufacturer’s instructions. The samples were sequenced using a 2x150 Pair-End (PE) configuration.

treatment_protocol: For codY- and covRS-deletion mutants and their controls, cells were harvested at their respective glucose-maltose diauxix shift. For comX overepression and its control, cells were harvested at mid log phase.

tag: strain

tag: genetic modification

value: comX overexpression vector

value: PxylT-comX

value: mid log phase

value: bacterial cell


title: Blood,excercised,cell611,24M1


title: WT_M17X

value: empty vector

value: WT

tag: treatment


title: CovRS_CDM

value: covRS deletion

value: DcovRS

value: diauxic shift


title: Blood,excercised,cell609,24M1


title: WT_CDM


title: Bulk BCR, adult male, subject P05, PBMC, year 2 day-6

description:
P05_BCR.tsv
P05_PBMC-ASC_d6_Y2


title: WT_CDMDEB


title: CodY_CDMDEB

value: codY deletion

value: DcodY


title: Bulk BCR, adult male, subject P04, PBMC, year 1 day-5

description:
P04_BCR.tsv
P04_PBMC-ASC_d5_Y1


title: Blood,excercised,cell608,24M1


title: Blood,excercised,cell606,24M1


title: Blood,excercised,cell604,24M1


title: Blood,excercised,cell605,24M1


title: TT_4h_washout_BRM014_rep2

city: Boston

first: Julia

middle: M

last: Rogers

state: Massachusetts

address: 45 Shattuck Street

department: BCMP

institute: Harvard Medical School

description:
sorted_4h_washout_BRM014_merged_depthnormalized_plus_single.bedGraph.bw
sorted_4h_washout_BRM014_merged_depthnormalized_minus_single.bedGraph.bw

data_processing:
*library strategy: Transient transcriptome sequencing (TT-seq)
Read pairs were trimmed to 100 base pairs using a custom script (trim_and_filter_PE.pl, https://doi.org/10.5281/zenodo.11222086), and reads with a minimum average quality score of 20 were kept. Reads were first mapped to the spike genome (dm6) using bowtie1.2.2 (-n2 -l 40 -X1000 -p 5 --best -3 1 parameters). Reads not mapping to the spike genome were mapped to the human genome (hg38) using with STAR2.7.3a40, using parameters --quantMode TranscriptomeSAM GeneCounts --outSAMtype BAM SortedByCoordinate --limitBAMsortRAM 42949672960 --outMultimapperOrder Random --outSAMattrIHstart 0 --outFilterType BySJout --outFilterMismatchNmax 4 --alignSJoverhangMin 8 --outSAMstrandField intronMotif --outFilterIntronMotifs RemoveNoncanonicalUnannotated --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --outWigType bedGraph --outWigNorm None --outFilterScoreMinOverLread 0 –outFilterMatchNminOverLread 0. Duplicates were also removed using STAR, and stranded coverage bedGraph files were generated from deduplicated BAM files using STAR. To normalize samples, reads mapping to exons in the active gene models  were counted using featurecounts, and used to calculate size factors using DESeq2. These size factors were scaled so the minimum was 1, and used to normalize bedGraphs with the custom script normalize_bedGraph.pl. Biological replicates (n=2) were merged using the script bedgraphs2stbedGraph.pl.
Assembly: hg38
Supplementary files format and content: bigwig files with replicates merged for each time point
Supplementary files format and content: excel file with read counts per gene for all samples
Library strategy: TT-seq

molecule: nuclear RNA

growth_protocol: SC2 squamous cell carcinoma cells were cultured in keratinocyte media, consisting of: 3:1 DMEM:F12 + 10% Fetal Bovine Serum (Gemini Bio-Products 100-106) + 1X Penicillin-Streptomycin (ThermoFisher 15140163) + 1X Reagent Mix (see below) + 1 μM Compound E (EMD Millipore565790) gamma secretase inhibitor (GSI). 100X Reagent Mix was prepared in 3:1 DMEM:F12 media, containing 40 mg/mL Hydrocortisone (Sigma-Aldrich H0888), 500 ng/mL Insulin (Sigma-Aldrich I6634), 1mg/mL EGF (Sigma-Aldrich E9644), 0.84 mg/mL Cholera toxin (Sigma-Aldrich C9903), 500 mg/mL Transferrin (Sigma-Aldrich T2036), and 1.3 mg/mL Liothyronine (Sigma-Aldrich T6397). GSI was prepared as a 1 mM stock of Compound E in DMSO (Sigma-Aldrich 472301). Cells were split every 3-4 days, using 0.25% trypsin (VWR 45000-664) supplemented with 1 μM GSI. Cells were incubated in 0.25% trypsin at 37 ºC until cells began to slough off the plate, then were quenched with fresh media and replated. Cells were routinely tested for mycoplasma, and used for experiments within 1 month of thawing a fresh vial.

extract_protocol:
Washout and mock washouts were performed as described above in the GSI washout section. 1 µM BRM014 or the equivalent volume of DMSO was added in the replenishing media after the two media changes. Labeling was performed for 10 minutes in 15 mL of media supplemented with 500 µM 4-Thiouridine (4-SU) (Thermo Fisher J60679) with GSI and/or BRM014 depending on the experimental condition. For the 0 h timepoints, labeling media was added immediately after the mock washouts. For the 1 hr and 4 hr timepoints, labeling media was added at 50 min or 3 h and 50 min after washout, respectively. To harvest cells for TT-seq, plates were quickly rinsed with 20 mL PBS; then 2 mL of Trizol (Thermo Scientific15596026) was added to the plate. After 3 min of lysis in the dish, the cell lysate in Trizol was collected and frozen at -80°C. 1.4 mL of each sample was used to prepare RNA. For normalization purposes, fly spike-in cells were used. Fly (S2) cells used for spike-in were labeled for 2 hr with 4-SU (500 µM), and harvested in Trizol. 5 % 4-SU labeled spike-in cells (e.g. 0.5 million fly cells into 10 million human cells) were added directly to the Trizol lysate for each experimental condition. This was then split into two 700 µL tubes for purification. First the lysate was homogenized using a QiaShredder column (Qiagen 79656). 150 µL of chloroform was added to the homogenate, which was then vortexed, incubated for 5 minutes, and spun down at 12,000 g for 15 minutes at 4ºC to separate the phases. 360 μL of the aqueous phase was transferred to a new tube, and 540 µL of ethanol with 1.5 mM DTT was added. This material was then applied to miRNeasy mini kit column (Qiagen 217004), and RNA was isolated following the kit protocol, using an on-column digestion with DNase (Qiagen 79254). After the isolation, the two columns per sample were re-combined. Aliquots of RNA were removed for quantification by spectrophotometry and analysis of RNA integrity by Agilent TapeStation 4200 using RNA high sensitivity tapes. All samples were confirmed to have RNA integrity numbers (RIN) > 9.0 before proceeding to TT-seq library construction.
50 µg of RNA per sample was brought to a volume of 80 µL with nuclease-free water and placed on ice. RNA was then lightly fragmented by addition of 20 µL cold 5X fragmentation solution (375 mM Tris-HCl (pH 8.3), 562.5 mM KCl, 22.5 mM MgCl2) and incubation at 94°C for 2 minutes 15 seconds. At the end of the fragmentation time, RNA was placed immediately on ice and 25 µL of cold 250 mM EDTA was added. RNA was precipitated by addition of 1/10 volume of 5 M NaCl, 2.5 volumes of 100 % ethanol and incubation at -20°C overnight. RNA was pelleted, washed, quantified, and analyzed again as above. Fragmented RNA was biotinylated essentially as described in Duffy et al.36 with the following modifications: the biotinylation reaction was performed in a total volume of 200 µL and allowed to incubate for 45 min in the dark. Excess biotin was removed using chloroform:isoamyl alcohol as per Dölken et al.37 and MaXtract tubes (Qiagen 129056) were used to separate organic and aqueous phases. Biotinylated RNA was resuspended in 100 µL of nuclease-free water and aliquots taken to use as the total RNA input fraction. In parallel, Dynabeads M-280 Streptavidin (ThermoFisher 11205D) were prepared for binding to render them RNase-free: for each sample, 75 µL of beads were used and treated in batch to render them RNase free. The beads were incubated 10min in a solution of 100 mM NaOH, 50 mM NaCl, placed on a magnetic stand, and then washed, resuspending the beads fully for each wash, twice with 500 µL of 100 mM NaCl, twice with 1 X TT-seq wash solution (100 mM Tris-HCl (pH 7.5), 10mM EDTA, 1 M NaCl, 0.05% Tween 20 in nuclease free water to which 1 µL SUPERase-In RNase Inhibitor (ThermoFisher AM2696) per 5mL solution is added prior to use), once in 0.3 X TT-seq wash solution, and finally resuspended in 52 µL/sample of 0.3 X TT-seq wash solution + 1 µL/sample SuperaseIn RNase Inhibitor.

treatment_protocol: For all gamma secretase inhibitor (GSI) washouts, media was removed, cells were washed twice with media containing 0.1% DMSO instead of GSI, and replenished with media containing 0.1%  DMSO instead of GSI, for a total of three media changes. In the mock washouts, both the washing media and the replenishing media contained 1 μM GSI. For experiments with BRM014 (MedChem Express HY-119374), the replenishing media, but not the washout media, either contained 1µM BRM014 or 0.01% DMSO depending on the sample. The evening before the time course, 11 15-cm dishes were seeded with 10 million SC2 cells per plate in SC2 media with 1 µM GSI. For each time course replicate (n=2), 10 samples were prepared: 0 hr mock washout + BRM014 (MedChem Express), 0 hr mock washout – BRM014 (reference condition in this text), 1 hr washout + BRM014, 1 hr washout - BRM014, 1 hr mock washout + BRM014, 1 hr mock washout - BRM014, 4 hr washout + BRM014, 4 hr washout - BRM014, 4 hr mock washout + BRM014, 4 hr mock washout - BRM014. The 11th plate was used as a sentinel plate: cells were harvested from the plate using 0.25% trypsin  and were counted to determine the total number of cells per plate. For each replicate, it was assumed that all 10 experimental plates had the same number of cells as the sentinel plate. Replicates were performed on different days (n=2).

value: squamous cell carcinoma

value: GSI washout


title: TT_4h_GSI_DMSO_rep2

description:
sorted_4h_GSI_DMSO_merged_depthnormalized_plus_single.bedGraph.bw
sorted_4h_GSI_DMSO_merged_depthnormalized_minus_single.bedGraph.bw

value: mock washout


title: TT_4h_washout_DMSO_rep1

description:
sorted_4h_washout_DMSO_merged_depthnormalized_plus_single.bedGraph.bw
sorted_4h_washout_DMSO_merged_depthnormalized_minus_single.bedGraph.bw


title: TT_4h_washout_BRM014_rep1


title: TT_4h_washout_DMSO_rep2


title: Blood,excercised,cell601,24M1

value: Blood

value: human samples

value: Memory B cells


title: Blood,excercised,cell602,24M1


title: TT_4h_GSI_DMSO_rep1


title: Blood,excercised,cell599,24M1


title: Blood,excercised,cell600,24M1


title: TT_4h_GSI_BRM014_rep1

description:
sorted_4h_GSI_BRM014_merged_depthnormalized_plus_single.bedGraph.bw
sorted_4h_GSI_BRM014_merged_depthnormalized_minus_single.bedGraph.bw


title: TT_1h_washout_DMSO_rep2

description:
sorted_1h_washout_DMSO_merged_depthnormalized_plus_single.bedGraph.bw
sorted_1h_washout_DMSO_merged_depthnormalized_minus_single.bedGraph.bw


title: Blood,excercised,cell598,24M1


title: Blood,excercised,cell597,24M1


title: TT_1h_washout_DMSO_rep1


title: TT_1h_washout_BRM014_rep2

description:
sorted_1h_washout_BRM014_merged_depthnormalized_plus_single.bedGraph.bw
sorted_1h_washout_BRM014_merged_depthnormalized_minus_single.bedGraph.bw


title: Blood,excercised,cell607,24M1


title: Blood,excercised,cell596,24M1


title: Blood,excercised,cell610,24M1


title: TT_1h_washout_BRM014_rep1


title: Blood,excercised,cell595,wk04th_infec


title: Blood,excercised,cell603,24M1


title: Blood,excercised,cell594,wk04th_infec


title: Blood,excercised,cell592,wk04th_infec


title: TT_1h_GSI_BRM014_rep1

description:
sorted_1h_GSI_BRM014_merged_depthnormalized_plus_single.bedGraph.bw
sorted_1h_GSI_BRM014_merged_depthnormalized_minus_single.bedGraph.bw


title: Blood,excercised,cell591,wk04th_infec


title: TT_0h_GSI_BRM014_rep2

description:
sorted_0h_GSI_BRM014_merged_depthnormalized_plus_single.bedGraph.bw
sorted_0h_GSI_BRM014_merged_depthnormalized_minus_single.bedGraph.bw


title: TT_0h_GSI_DMSO_rep2

description:
sorted_0h_GSI_DMSO_merged_depthnormalized_plus_single.bedGraph.bw
sorted_0h_GSI_DMSO_merged_depthnormalized_minus_single.bedGraph.bw


title: TT_4h_GSI_BRM014_rep2


title: Blood,excercised,cell590,wk04th_infec


title: TT_0h_GSI_BRM014_rep1


title: TT_0h_GSI_DMSO_rep1


title: Blood,excercised,cell588,wk04th_infec


title: Blood,excercised,cell587,wk04th_infec


title: Blood,excercised,cell586,wk04th_infec


title: PRO_4h_rep1

description:
4h_F.bw
4h_R.bw

data_processing:
Read pairs were trimmed using cutadapt 1.14 to remove adapter sequences (-O 1 --match-read-wildcards -m {20}). An additional nucleotide was removed from the end of read 1 (R1), using seqtk trimfq (https://github.com/lh3/seqtk), to preserve a single mate orientation during alignment. The paired end reads were then mapped to a combined genome index, including both the spike (dm6) and primary (hg38) genomes, using bowtie242. Properly paired reads were retained. These read pairs were then separated based on the genome (i.e. spike-in vs primary) to which they mapped. Reads mapping to the reference genome were separated according to whether they were R1 or R2, sorted via samtools43 1.3.1 (-n), and subsequently converted to bedGraph format using a custom script (bowtie2stdBedGraph.pl https://doi.org/10.5281/zenodo.11222086). We note that this script counts each read once at the exact 3’ end of the nascent RNA. Because R1 in PRO-seq reveals the position of the RNA 3’ end, the “+” and “-“ strands were swapped to generate bedGraphs representing 3’ end positions at single nucleotide resolution. As spike percentages were not significantly different between samples, biological replicates were depth normalized using the script normalize_bedGraph.pl. Biological replicates (n=3 for t=0, n=2 for all other time points) were merged using the script bedgraphs2stbedGraph.pl. The merged t=0 bedgraph was normalized by multiplying by 0.66 to using normalize_bedGraph.pl so that all time points had an equivalent depth. BedGraph files were binned in 10 bp intervals, and converted to bigwig files for visualization on the genome browser using UCSCtools.
Assembly: hg38
Supplementary files format and content: bigwig files with replicates merged for each time point
Library strategy: PRO-seq

extract_protocol:
To permeabilize cells, at the relevant time post washout, plates were rinsed with PBS, and 0.25 % trypsin (containing GSI for the mock washout, or an equivalent amount of DMSO for all other time points) was added. Once cells were detaching from the plate, trypsin was quenched with cold SC2 media (containing GSI for the mock washout, or an equivalent amount of DMSO for all other time points), and cells were transferred to a 15 mL falcon tube. Cells were pelleted at 300 g for 4 min at 4°C, supernatant was removed, and cells were resuspended in 10 mL cold PBS. Again, cells were pelleted at 300 g for 4 min at 4°C, supernatant was removed, and cells were resuspended in 250 µL cold Buffer W (10 mM Tris-Cl, pH 8.0, 10 mM KCl, 250 mM sucrose, 5 mM MgCl2, 1 mM EGTA, 10% (w/v) glycerol in DEPC ddH2O + 1 protease inhibitor tablet (Millipore Sigma 5056489001), 0.5 mM DTT,  and 10 µL SUPERase-In RNase inhibitor per 50 mL buffer added fresh before beginning the experiment). 10 mL of Buffer P (10 mM Tris-Cl, pH 8.0, 10 mM KCl, 250 mM sucrose, 5 mM MgCl2, 1 mM EGTA, 0.05 % Tween-20 10 % (w/v) glycerol in DEPC ddH2O + 1 protease inhibitor tablet, 0.5 mM DTT, and 10 µL SUPERase-In RNase inhibitor per 50 mL buffer added fresh before beginning the experiment) was slowly added to the tube, and cells were incubated for 5 minutes on ice to permeabilize. Two times, cells were pelleted at 400 g for 4 min at 4°C, supernatant was removed, and cells were resuspended in 1 mL Buffer W, and pipetted up and down slowly with a wide-bore pipette tip. An additional 9 mL of Buffer W was added. After the second wash, cells were pelleted at 500 g for 4 min at 4°C, resuspended in 250 L freezing buffer (10 mM Tris-Cl pH 8, 40% (w/v) glycerol, 5 mM MgCl2, 1.1 mM EDTA + 1 protease inhibitor tablet, 0.5 mM DTT, and 10 L SUPERase-In RNase inhibitor per 50 mL buffer added fresh before beginning the experiment), and moved to a 1.5 mL tube. The 15 mL tube was rinsed with another 250 L freezing buffer, which was combined to make 500 µL of permeabilized cells in freezing buffer, which were snap frozen in liquid nitrogen and stored at -80°C.
PRO-seq library construction was performed by the Nascent Transcriptomics Core at Harvard Medical School, Boston, MA. Aliquots of frozen permeabilized cells were thawed on ice and pipetted gently to fully resuspend. Aliquots were removed and permeabilized cells were counted using a Luna II, Logos Biosystems instrument. For each sample, 1 million permeabilized cells were used for nuclear run-on, with 50,000 permeabilized Drosophila S2 cells added to each sample for normalization. Nuclear run-on assays and library preparation were performed essentially as described in Reimer et al. with modifications noted: 2X nuclear run-on buffer consisted of (10 mM Tris (pH 8), 10 mM MgCl2, 1 mM DTT, 300 mM KCl, 20 mM/each biotin-11-NTPs (Perkin Elmer NEL54(2/3/4/5)001), 0.8 U/mL SuperaseIN, 1 % sarkosyl (Sigma-Aldrich L7414).  Run-on reactions were performed at 37°C.  Fragmentation of RNA was performed in 100 mM NaOH at 0°C for 10 minutes and stopped by addition of one volume of ice cold 1 M Tris (pH 6.8).  Adenylated 3' adapter was prepared using the 5' DNA adenylation kit (New England Biolabs E2610S) and ligated using T4 RNA ligase 2, truncated KQ (NEB, per manufacturer’s instructions with 15 % PEG-8000 final) and incubated at 16°C overnight.  180 mL of betaine blocking buffer (1.42 g of betaine brought to 10 mL with binding buffer supplemented to 0.6 mM blocking oligo (TCCGACGATCCCACGTTCCCGTGG/3InvdT/)) was mixed with ligations and incubated 5 min at 65°C and 2 min on ice prior to addition of Dynabead M-280 streptavidin beads (ThermoFisher 11205D). After T4 polynucleotide kinase (New England Biolabs M0201) treatment, beads were washed once each with high salt, low salt, and blocking oligo wash (0.25X T4 RNA ligase buffer (New England Biolabs M0437), 0.3 mM blocking oligo) solutions and resuspended in 5' adapter mix (10 pmol 5' adapter, 30 pmol blocking oligo, water).  5' adapter ligation was per Reimer et al.38, but with 15 % PEG-8000 final. Eluted cDNA was amplified 5-cycles (NEBNext Ultra II Q5 master mix (New England Biolabs M0544) with Illumina TruSeq PCR primers RP-1 and RPI-X) following the manufacturer's suggested cycling protocol for library construction. A portion of preCR was serially diluted and for test amplification to determine optimal amplification of final libraries. Pooled libraries were sequenced using the Illumina 200 cycle kit on an S4 single lane, followed by additional run on a full SP lane for more depth for some samples.

treatment_protocol: For all gamma secretase inhibitor (GSI) washouts, media was removed, cells were washed twice with media containing 0.1% DMSO instead of GSI, and replenished with media containing 0.1%  DMSO instead of GSI, for a total of three media changes. In the mock washouts, both the washing media and the replenishing media contained 1 μM GSI. The evening before the time course, cells were plated on 9 10-cm dishes, at 5.5 million cells / plate in SC2 media with 1 µM GSI. For each time course, 9 samples were prepared: 0 hr mock washout, 15 min washout, 30 min washout, 45 min washout, 1 hr washout, 90 min washout, 2 hr washout, 3 hr washout, and 4 hr washout. Replicates were performed on different days (n=2, n=3 for the reference condition).

tag: time


title: PRO_4h_rep2

description:
4h_F.bw
4h_R.bw


title: PRO_2h_rep2

description:
2h_F.bw
2h_R.bw


title: TT_1h_GSI_DMSO_rep2

description:
sorted_1h_GSI_DMSO_merged_depthnormalized_plus_single.bedGraph.bw
sorted_1h_GSI_DMSO_merged_depthnormalized_minus_single.bedGraph.bw


title: Blood,excercised,cell584,wk04th_infec


title: Blood,excercised,cell582,wk04th_infec


title: PRO_3h_rep1

description:
3h_F.bw
3h_R.bw


title: Blood,excercised,cell583,wk04th_infec


title: PRO_3h_rep2

description:
3h_F.bw
3h_R.bw


title: Blood,excercised,cell585,wk04th_infec


title: TT_1h_GSI_BRM014_rep2


title: Blood,excercised,cell593,wk04th_infec


title: TT_1h_GSI_DMSO_rep1


title: Blood,excercised,cell581,wk04th_infec


title: Blood,excercised,cell580,wk04th_infec


title: PRO_90min_rep2

description:
90m_F.bw
90m_R.bw


title: Blood,excercised,cell579,wk04th_infec


title: Blood,excercised,cell589,wk04th_infec


title: PRO_90min_rep1

description:
90m_F.bw
90m_R.bw


title: PRO_1h_rep2

description:
1h_F.bw
1h_R.bw


title: Blood,excercised,cell578,wk04th_infec


title: PRO_1h_rep1

description:
1h_F.bw
1h_R.bw


title: PRO_45min_rep2

description:
45m_F.bw
45m_R.bw


title: Blood,excercised,cell577,wk04th_infec


title: Blood,excercised,cell576,wk04th_infec


title: Blood,excercised,cell575,wk04th_infec


title: PRO_45min_rep1

description:
45m_F.bw
45m_R.bw


title: PRO_30min_rep2

description:
30m_F.bw
30m_R.bw


title: Blood,excercised,cell573,wk04th_infec


title: PRO_30min_rep1

description:
30m_F.bw
30m_R.bw


title: Blood,excercised,cell574,wk04th_infec


title: PRO_2h_rep1

description:
2h_F.bw
2h_R.bw


title: PRO_15min_rep3

description:
15m_F.bw
15m_R.bw


title: Blood,excercised,cell571,wk04th_infec


title: Blood,excercised,cell570,wk04th_infec


title: Blood,excercised,cell569,wk04th_infec


title: Blood,excercised,cell567,wk04th_infec


title: H3K27ac_ChIP_4h_input_repB

description: 4h_input_depthnorm_50bp_75shift_merge.bedgraph.bw

data_processing:
Read 1 was trimmed to positions 2-49 using a custom script (trim_and_filter_SE.pl, https://doi.org/10.5281/zenodo.11222086), and reads with a minimum average quality score of 20 were kept. Reads were mapped first to the spike genome (dm6), then to the human genome (hg38) using bowtie1.2.245 with options -k1 -v2 – best. The custom script bowtie2stdBedGraph.pl was used to make bedGraph files with a 75bp shift. As spike returns were not different between time points, samples were depth normalized using the script normalize_bedGraph.pl. Biological replicates (n=2) were merged using the script bedgraphs2stbedGraph.pl
Assembly: hg38
Supplementary files format and content: bigwig files with replicates merged for each time point

extract_protocol:
To crosslink cells, at the appropriate time after washout, media was removed from the plates and 10 mL PBS was added (PBS contained 1 mM GSI for the mock washout sample). 9 mL of 2.1 % formaldehyde in PBS was added to the plate (1% final concentration) (3 plates / sample were staggered by 15 seconds to ensure equal crosslinking time), and cells were fixed on a shaker for precisely 15 min. 1 mL 2.5 M Glycine was added after 15 min, plates were incubated for an additional 5 min, and then placed on ice. Cells were scraped off the plate and collected in a 50 mL falcon tube. Plates were rinsed with 20 mL cold PBS, which was added to the falcon tube. Cells were pelleted at 300 g for 5 min at 4°C, supernatant was discarded. The 3 tubes/sample were combined and resuspended in 40 mL of PBS, pelleted at 300 g for 5 min at 4°C, resuspended in 10 mL PBS, and pelleted again. The pellet was then resuspended in 250 mL of sonication buffer (20 mM Tris (pH 8.0), 2 mM EDTA, 0.5 mM EGTA, 0.5 % SDS, 0.5 mM PMSF, and 1 mini protease inhibitor tablet per 10 mL buffer. Chromatin was incubated on ice for 10min, 2 5 mL samples were taken for chromatin normalization, and then samples were flash frozen and stored at -80°C. To normalize chromatin, the 5 mL sample was added to 200 mL QuickExtract Buffer (VWR). Samples were vortexed 15 s, heated at 65°C at 1500 rpm for 15 min, vortexed again for 15 s, then heated at 98°C for 6 minutes. A standard sample of SC2 cells at 108 cells/mL was processed in this same way to make a standard. The DNA concentration of all samples and the standards were measured by the Qubit dsDNA high sensitivity quantitation kit (ThermoFisher), and samples were adjusted to the 108 cells/mL standard with fresh sonication buffer. Chromatin was then sonicated using a QSonica sonicator (70 % amplitude, 15 seconds ON / 45 seconds off cycles) to an average fragment size of ~200 bp, flash frozen, and stored at -80°C. Chromatin from 2.5 million cells was diluted in 1 mL IP buffer (20 mM Tris (pH 8.0), 2 mM EDTA, 0.5% Triton X-100, 150 mM NaCl, 10% Glycerol), and precleared for 2 hours at 4°C, rotating with 30 µL Protein A agarose beads (Millipore Sigma 16-125) that had been pre-equilibrated in cold IP buffer.  Precleared input was transferred to a new tube, 250 µL additional IP buffer and 30 µL H3K27ac antibody (Active Motive 39133) were added, and samples were incubated overnight at 4°C, rotating. 200 µL Protein A beads were added to each IP, and samples were incubated for 2 hours at 4°C, rotating. Samples were washed once with Low-Salt Buffer (20 mM Tris (pH 8.0), 2 mM EDTA, 1% Triton X-100, 150 mM NaCl, 0.1% SDS), three times with High- Salt Buffer (20 mM Tris pH 8.0, 2 mM EDTA, 1% Triton X-100, 500 mM NaCl, 0.1% SDS), once with Lithium Chloride Buffer (20 mM Tris pH 8.0, 2 mM EDTA, 250 mM LiCl, 1% IGEPAL CA- 630, 1% sodium deoxycholate), and twice with TE Buffer (10 mM Tris-HCl, 1 mM EDTA). For each wash, samples were rotated for 3 minutes with 1 mL of cold wash solution. Samples were eluted twice by rotating beads for 15 min at room temperature with 250 µL elution buffer (1 % SDS, 100 mM NaHCO3). The two elutions were combined, NaCl was added to 200 mM, and samples were incubated overnight at 65°C to reverse crosslinks. Samples were treated with Proteinase K (New England Biolabs P8107) for 30 min, extracted with phenol:chloroform:isoamyl alcohol, and resuspended in 100 µL water.
For spike normalization, the same amount of sheared Drosophila DNA was added to each sample before library construction. Libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs E7645) and sequenced paired end on a NovaSeq with an S1 single lane 100 cycle kit.

treatment_protocol: For all gamma secretase inhibitor (GSI) washouts, media was removed, cells were washed twice with media containing 0.1% DMSO instead of GSI, and replenished with media containing 0.1%  DMSO instead of GSI, for a total of three media changes. In the mock washouts, both the washing media and the replenishing media contained 1 μM GSI. Two days before the time course, cells were plated 1:4 into three 15 cm dishes per sample in SC2 media with 1 µM GSI. For each time course, 8 samples were prepared: 0 hr mock washout, 15 min washout, 30 min washout, 45 min washout, 1 hr washout, 90 min washout, 2 hr washout, and 4 hr washout. Replicates were performed on different days (n=2).


title: Blood,excercised,cell568,wk04th_infec


title: PRO_ref_rep1

description:
0m_F.bw
0m_R.bw


title: Blood,excercised,cell572,wk04th_infec


title: Blood,excercised,cell566,wk04th_infec


title: PRO_15min_rep2

description:
15m_F.bw
15m_R.bw


title: Blood,excercised,cell565,wk04th_infec


title: H3K27ac_ChIP_2h_ repB

description: 2h_depthnorm_50bp_75shift_merge.bedgraph.bw


title: H3K27ac_ChIP_4h_ repA

description: 4h_depthnorm_50bp_75shift_merge.bedgraph.bw


title: PRO_ref_rep3

description:
0m_F.bw
0m_R.bw


title: Blood,excercised,cell564,wk04th_infec


title: H3K27ac_ChIP_2h_ repA


title: Blood,excercised,cell563,wk04th_infec


title: H3K27ac_ChIP_90m_repB

description: 90m_depthnorm_50bp_75shift_merge.bedgraph.bw


title: Blood,excercised,cell562,wk04th_infec


title: Blood,excercised,cell561,wk04th_infec


title: H3K27ac_ChIP_4h_ repB


title: Blood,excercised,cell560,wk04th_infec


title: Blood,excercised,cell559,wk04th_infec


title: H3K27ac_ChIP_90m_repA


title: PRO_ref_rep2

description:
0m_F.bw
0m_R.bw


title: H3K27ac_ChIP_1h_repA

description: 1h_depthnorm_50bp_75shift_merge.bedgraph.bw


title: Blood,excercised,cell558,wk04th_infec


title: H3K27ac_ChIP_45m_repB

description: 45m_depthnorm_50bp_75shift_merge.bedgraph.bw


title: H3K27ac_ChIP_4h_input_repA


title: H3K27ac_ChIP_45m_repA


title: Blood,excercised,cell557,wk04th_infec

description: SMARTseq2 single cell RNA sequencing data from E2-specific memory B cells


title: H3K27ac_ChIP_30m_repA

description: 30m_depthnorm_50bp_75shift_merge.bedgraph.bw


title: Blood,excercised,cell554,wk04th_infec


title: Blood,excercised,cell556,wk04th_infec


title: Blood,excercised,cell555,wk04th_infec


title: H3K27ac_ChIP_15m_repB

description: 15m_depthnorm_50bp_75shift_merge.bedgraph.bw


title: H3K27ac_ChIP_30m_repB


title: Blood,excercised,cell553,wk04th_infec


title: Blood,excercised,cell552,wk04th_infec


title: H3K27ac_ChIP_15m_repA


title: H3K27ac_ChIP_0h_input_repB

description: 0h_input_depthnorm_50bp_75shift_merge.bedgraph.bw


title: Blood,excercised,cell551,wk04th_infec


title: Blood,excercised,cell548,wk04th_infec


title: Blood,excercised,cell550,wk04th_infec


title: Blood,excercised,cell549,wk04th_infec


title: H3K27ac_ChIP_1h_repB


title: ATAC_4h_washout_DMSO_rep2

description: merged_ATAC_single_4_washout_dmso.bedGraph.bw

data_processing:
Read pairs were trimmed to 100 base pairs using a custom script (trim_and_filter_PE.pl, https://doi.org/10.5281/zenodo.11222086), and reads with a minimum average quality score of 20 were kept. Adapter sequences were removed using cutadapt 1.1441. Reads were first mapped to the spike genome (dm6), using bowtie1.2.2 (-k1 -v2 -X1000 – allow-contain -- best parameters). Reads that did not map to the spike genome were mapped to the human genome (hg38), using bowtie1.2.245 (-k1 -v2 -X1000 – allow-contain -- best parameters). Samtools 1.943 was used to flag duplicate reads, which were then removed. A custom script, extract_fragments.pl, was used to filter and retain unique reads between 10 and 150 base pairs, which correspond to regions of open chromatin, and convert files into bedGraph format. Spike return rates were not significantly different between samples. To normalize samples, reads mapping to the -1 kb to +1 kb region around active promoters in this cell type were counted using the custom script makeheatmap with the “-b v -v t -s b” options. The number of these reads was used to calculate size factors using DESeq246. These size factors were scaled so the minimum was 1, and used to normalize bedGraphs with the custom script normalize_bedGraph.pl. Biological replicates (n=2) were merged using the script bedgraphs2stbedGraph.pl.
Assembly: hg38
Supplementary files format and content: bigwig files with replicates merged for each time point

extract_protocol:
At the time point after washout or mock washout, cells were rinsed in PBS, and harvested using 0.25 % trypsin with GSI and/or BRM014 according to the sample, and quenched with SC2 media containing GSI and/or  BRM014. Cells were counted, and 100,000 cells were transferred to an Eppendorf tube, and combined with 10,000 fly spike-in cells. The spike-in S2 cells had been collected, resuspended in BAMBANKER cryopreservative media (VWR 101974-112), aliquoted, and stored at -80°C. All spike in aliquots for one experimental time course replicate were thawed and combined before beginning the time course. ATAC was performed according to the OmniATAC protocol. Briefly, cells were pelleted at 500 g for 5 min at 4°C, and supernatant was removed. The cell pellet was resuspended in 50 mL ATAC lysis buffer by pipetting up and down 3 times (Cold ATAC-Resuspension Buffer (10 mM Tris–HCl pH 7.5, 10 mM NaCl and 3 mM MgCl2) + 0.1 % NP40 (EMD Millipore 492018) + 0.1 % Tween-20 (EMD Millipore 655206) + 0.01 % digitonin (Promega G9441)). Up to 4 samples were processed at 1 time, and samples were staggered by 15 seconds. After exactly 3 min of lysis, 1 mL of ATAC wash buffer (Cold ATAC-Resuspension Buffer + 0.1 % Tween-20 (EMD Millipore655206)) was added to the tube. Nuclei were pelleted at 500 g for 10 min at 4°C, and supernatant was removed. Nuclei were resuspended in 50 mL of Transposition mix (1x TD buffer (Illumina 20334197), 3 mL Tn5 transposase (Illumina 20334197), 0.33x PBS, 0.01 % digitonin, 0.1 % Tween-20), and incubated at 37°C in a thermomixer at 1000 rpm for 30 minutes. The reactions were quenched with 250 mL of binding buffer from the MinElute PCR purification kit (Qiagen 28004) and purified using this kit.
The eluate was then used as the input for barcoding PCR, to add sequencing adapters and sequencing barcodes, as described in Grandi et al.. Both the qPCR and Qubit methods were used to quantify the libraries to determine the total number of PCR cycles, as described in the OmniATAC protocol. After the final PCR cycles, libraries were cleaned up with the MinElute PCR purification kit, quantified using the NEBNext Library Quant kit (New England Biolabs E7630), and pooled for sequencing. Libraries were sequenced paired end on a NovaSeq with an S1 single lane 100 cycle kit.

treatment_protocol: For all gamma secretase inhibitor (GSI) washouts, media was removed, cells were washed twice with media containing 0.1% DMSO instead of GSI, and replenished with media containing 0.1%  DMSO instead of GSI, for a total of three media changes. In the mock washouts, both the washing media and the replenishing media contained 1 μM GSI. For experiments with BRM014 (MedChem Express HY-119374), the replenishing media, but not the washout media, either contained 1µM BRM014 or 0.01% DMSO depending on the sample. The evening before the time course, cells were plated in 12 well dishes, at 125,000 cells / well in SC2 media with 1 µM GSI. For each time course, 14 samples were prepared: 0 hr mock washout + BRM014, 0 hr mock washout – BRM014, 1 hr washout + BRM014, 1 hr washout - BRM014, 1 hr mock washout + BRM014, 1 hr mock washout - BRM014, 2 hr washout + BRM014, 2 hr washout - BRM014, 2 hr mock washout + BRM014, 2 hr mock washout - BRM014, 4 hr washout + BRM014, 4 hr washout - BRM014, 4 hr mock washout + BRM014, 4 hr mock washout - BRM014. Replicates were performed on different days (n=2).


title: H3K27ac_ChIP_0h_repA

description: 0m_depthnorm_50bp_75shift_merge.bedgraph.bw


title: Blood,excercised,cell546,wk04th_infec


title: ATAC_4h_washout_BRM014_rep2

description: merged_ATAC_single_4_washout_brm014.bedGraph.bw


title: Blood,excercised,cell545,wk04th_infec


title: Blood,excercised,cell547,wk04th_infec


title: ATAC_4h_washout_DMSO_rep1


title: Blood,excercised,cell544,wk04th_infec


title: ATAC_4h_washout_BRM014_rep1


title: Blood,excercised,cell543,wk04th_infec


title: ATAC_4h_GSI_DMSO_rep1

description: merged_ATAC_single_4_gsi_dmso.bedGraph.bw


title: Blood,excercised,cell542,wk04th_infec


title: Blood,excercised,cell540,wk04th_infec


title: ATAC_4h_GSI_BRM014_rep2

description: merged_ATAC_single_4_gsi_brm014.bedGraph.bw


title: ATAC_4h_GSI_BRM014_rep1


title: Blood,excercised,cell541,wk04th_infec


title: Blood,excercised,cell539,wk04th_infec


title: H3K27ac_ChIP_0h_input_repA


title: ATAC_2h_washout_DMSO_rep2

description: merged_ATAC_single_2_washout_dmso.bedGraph.bw


title: H3K27ac_ChIP_0h_repB