title: Fixed scRNAseq,multiplexed pool 2

description: Library name: Pool_16_2

data_processing:
Demulitplexing and gene counting were performed by 10X CellRanger v7.0.0 using the Chromium Mouse Transcriptome Probe Set v1.0 for mm10. See feature_README.csv fpor demultiplexing strategy.
Assembly: mm10
Supplementary files format and content: Tab separated files corresponding to sample barcodes and features, and a matrix file containing raw gene count data for each sample.

extract_protocol:
ChATflox and ChATflox;Mb1-Cre mouse livers were collected at 2, 4, and 24 hours after PHX or Sham surgery, and 25mg of tissue from the same lobe across experimental samples were immediately snap-frozen.
Thawing and dissociation of cell samples were performed according to the Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling protocols provided by 10X Genomics. Sample probe hybridization, washing, pooling, and capture of approximately10,000 cells per sample, 4 samples per library (2 libraries), and followed by library construction, were performed according to 10X Genomics protocols for the 4-plex kit.

tag: multiplexed samples

value: Sequenced in 4-plex reaction. Sample 265 = BC001. Sample 267 = BC002. Sample 268 = BC004. Sample 269 = BC003. See feature_README.csv.


title: Nrf2_Bcat_TREAT_BL_567

status: Public on May 02 2025

type: SRA

city: Pittsburgh

first: Shuchang

last: Liu

state: PA

address: 203 Lothrop Street

country: USA

institute: University of Pittsburgh

library_source: transcriptomic

data_processing:
Low-quality reads and adapter sequences were filtered out by tool Trimmomatic
Reads were aligned to mouse reference genome mm10 by STAR aligner
Gene counts were quantified by STRA
Assembly: mm10
Supplementary files format and content: TAB

molecule: polyA RNA

organism: Mus musculus

source_name: liver

extract_protocol:
Using fresh frozen liver tissue, RNA was isolated using the RNeasy Mini kit (Qiagen) according to standard manufacturer protocols for tissue RNA isolation
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: tissue

value: liver

tag: treatment


title: ChAT-GFP positive liver B cells, 4 hours post-PHX, rep 6

city: Toronto

first: Liam

middle: D

last: Hendrikse

state: Ontario

address: 610 University Ave

department: Princess Margaret Cancer Centre

country: Canada

institute: University Health Network

description:
Library name: 7_S49
Column name in raw_RSEM_counts.txt: X7_S49

data_processing:
Adaptor content was trimmed using Trim Galore (v 0.6.6) on paired reads with default parameters.
Trimmed reads were then aligned to the mm10_STAR_2.7.9a mouse reference using STAR (v 2.7.9a).
Gene counts and transcripts per million (TPM) normalization were determined using RSEM (v 1.3.0).
Assembly: mm10
Supplementary files format and content: raw_RSEM_counts.txt: tab-delimited text file containing raw counts (*_counts), TPM normalized (*_tpm), and FPKM normalized (*_fpkm) values for all samples.
Supplementary files format and content: Sample_.genes.results: tab-delimited file containing RSEM gene alignment outputs for each sample.

molecule: total RNA

source_name: Liver

extract_protocol:
ChAT-GFP reporter mice were subjected to 70% PHX or Sham surgery and liver non-parenchymal cells were enriched as described above. B cells were stained and sorted on a BD FACSAria Fusion flow cytometry cell sorter at the Princess Margaret Flow Facility. RNA was extracted using the RNAeasy micro kit (QIAGEN) based on the manufacturer’s instructions.
Sample library preparation was performed using a Mammalian Low Input V2 Pico kit (Takara) based on the manufacturer’s instructions.

value: Liver

tag: Sex

value: Male

tag: cell type

value: B lymphocyte

tag: genotype

tag: mouse id

value: PHX

tag: fraction

value: GFP-Pos


title: Arabidopsis, Mock,HTA67, 12DAG, Rep2

city: Xianyang

first: Mingming

last: Zhao

address: Taicheng Road,Yangling District

country: China

institute: Northwest A&F University China

description: Library name: 12DAG_Mock_HTA67_Rep2,ChIP

library_source: genomic

data_processing:
(1)Filter:ChIP-seq reads were trimmed using the fastp tool (version 0.23.4)  with a quality score threshold of >20 for the final base.Reads shorter than 35 bp after trimming were discarded.(2)Alignment:The clean reads were aligned to the TAIR 10 genome (https://www.arabidopsis.org/) using Bowtie2 and sorted and indexed using the samtools suite (version 1.17).(3)Remove: PCR duplicates were removed using Picard (version 3.0.0). (4)Callpeak:Peaks were identified using the “callpeak” function of MACS2 (version 2.2.6,). (5)Coverage tracks were calculated: normalizing using RPGC (effective genome size 119482912) –bin size 10, normalization method using RPGC reads per genome coverage), creating bigwig files.(6)Plot:The metagene results were drawn using deepTools(version 3.5.2).
Assembly: TAIR 10
Supplementary files format and content: BigWig files were generated using the Deeptools  (version 3.5.2).The BigWig files for making plot were  assessed by merging results from 'bigwigcompare' of two independent biological replicates, employing the 'mean' operation.

molecule: genomic DNA

organism: Arabidopsis thaliana

source_name: 12DAG seedling

growth_protocol: Generally, plants were grown on soil mixture in a growth chamber under long-day conditions at ~21°C. Plants used for ChIP-Seq and TSA treatments were grown in MS media supplemented with 1% sucrose under long-day photoperiod and ~21°C. In all cases, seeds were stratified for 4 days at 4°C, and seedlings were harvested 12 days after germination.

extract_protocol:
1)Crosslink protein-DNA interactions with formaldehyde; 2) Fragment chromatin via sonication or enzymatic digestion; 3) Immunoprecipitate target protein-DNA complexes using specific antibodies; 4) Reverse crosslinking and purify DNA, followed by library construction for sequencing .
ChIP-Seq libraries were prepared using Ovation® Ultralow System V2 Library Preparation Kit (PART NO. 0344, 0344NB).

value: 12DAG seedling

tag: chip-antibody

value: CD771，杭州景杰


title: Nrf2_Bcat_CTRL_BL_568


title: ChAT-GFP positive liver B cells, sham surgery, rep 6

description:
Library name: 5_S47
Column name in raw_RSEM_counts.txt: X5_S47

value: Sham


title: Arabidopsis, Mock,HTA67, 12DAG, Rep1

description: Library name: 12DAG_Mock_HTA67_Rep1,ChIP


title: Nrf2_Bcat_TREAT_BL_566


title: Arabidopsis, Mock,H2A.Z, 12DAG, Rep1

description: Library name: 12DAG_Mock_H2A.Z_Rep1,ChIP


title: Arabidopsis, Mock,H2A.Z, 12DAG, Rep2

description: Library name: 12DAG_Mock_H2A.Z_Rep2,ChIP


title: Arabidopsis, TSA,H3, 12DAG, Rep2

description: Library name: 12DAG_TSA_H3_Rep2,ChIP


title: MDS C22.7 (high risk) R2

city: Birmingham

first: Peter

last: Keane

address: Vincent Drive

department: Institute for Cancer and Genomic Sciences

country: United Kingdom

institute: University of Birmingham

description: P3 100 cycle kit

data_processing:
Raw sequencing reads were processed to remove low-quality bases and Nextera ATAC adapter sequences using Trimmomatic v0.39 (Bolger et al. 2014; PMID:24695404)
Reads were then aligned to the human genome (version hg38) using Bowtie2 v2.2.5 (Langmead and Salzburg, 2012; PMID:22388286) with the --very-sensitive-local parameter.
Potential PCR duplicates were removed from the alignments using Picard MarkDuplicates v2.16.10 (http://broadinstitute.github.io/picard).
Peaks were called using MACS2 v2.2.7.1 (Zhang et al. 2008; PMID:18798982) with the options --nomodel --call-summits -q 0.05 -B --trackline.
Assembly: hg38
Supplementary files format and content: Read pileups in bedGraph format

organism: Homo sapiens

source_name: HSPCs CD34+CD45+

growth_protocol: Cells were grown in StemDiff (stemCell tehcnology) for 14 days

extract_protocol:
Freshly collected cells were collected and sort based on the positive expression of CD45 and CD34.
Fragment transposition ATAC libraries were generated following the Omni-ATAC protocol. doi: 10.1038/nmeth.4396

value: HSPCs CD34+CD45+

tag: risk category

value: High Risk

value: no treatment


title: ChAT-GFP negative liver B cells, sham surgery, rep 7

description:
Library name: 6_S48
Column name in raw_RSEM_counts.txt: X6_S48

value: GFP-Neg


title: Nrf2_Bcat_CTRL_BL_563


title: BL_551 LNP-CTRL

description:
Library name: Sample 1
BL_551 LNP-CTRL

data_processing:
10X Space Ranger was used to generate the gene by spot count matrix
Assembly: mm10
Supplementary files format and content: barcodes.tsv.gz: TSV file
Supplementary files format and content: features.tsv.gz: TSV file
Supplementary files format and content: matrix.mtx.gz: MTX file
Supplementary files format and content: H&E staining: TIF file
Library strategy: Spatial Transcriptomics

extract_protocol:
Tissue sections of 10um thickness were cut from mouse liver tumor FFPE blocks and were deparaffinized, H&E stained, then decrosslinked using standard 10X genomics protocols. Regions of interest (ROIs) were selected by outlining ~6.5mm2 areas.
Standard Visium CytAssist protocols were used for FFPE tissue for generation of sequence ready libraries. Quality control parameters were a DV200% >= 30%.

value: BL_551 LNP-CTRL


title: Nrf2_Bcat_CTRL_BL_562


title: ChAT-GFP negative liver B cells, 4 hours post-PHX, rep 6

description:
Library name: 4_S46
Column name in raw_RSEM_counts.txt: X4_S46


title: Arabidopsis, Mock,H3K27Ac, 12DAG, Rep2

description: Library name: 12DAG_Mock_H3K27Ac_Rep2,ChIP

value: 39135，Active Motif


title: Fixed scRNAseq,multiplexed pool 1

description: Library name: Pool_16_1

library_source: transcriptomic single cell

value: Sequenced in 4-plex reaction. Sample 258 = BC002. Sample 260 = BC001. Sample 277 = BC003. Sample 278 = BC004. See feature_README.csv.


title: ChAT-GFP negative liver B cells, sham surgery, rep 6

description:
Library name: 2_S44
Column name in raw_RSEM_counts.txt: X2_S44

value: Bcell


title: MDS C22.7 (high risk) R1

description: P3 100 cycle kit


title: Met_Bcat_TREAT_BL_183

value: liver


title: Arabidopsis, Mock,H3K27Ac, 12DAG, Rep1

description: Library name: 12DAG_Mock_H3K27Ac_Rep1,ChIP


title: Met_Bcat_TREAT_BL_182


title: ChAT-GFP negative liver B cells, 4 hours post-PHX, rep 5

description:
Library name: 27_S68
Column name in raw_RSEM_counts.txt: X27_S68

value: Liver


title: MDS C22 (low risk)  R2

description: P3 100 cycle kit

value: Low risk


title: MDS C22 (low risk)  R1

description: P3 100 cycle kit


title: ChAT-GFP positive liver B cells, 4 hours post-PHX, rep 4

description:
Library name: 26_S67
Column name in raw_RSEM_counts.txt: X26_S67

value: Male

value: B lymphocyte


title: Arabidopsis, Mock,H3K9K14Ac, 12DAG, Rep1

description: Library name: 12DAG_Mock_H3K9K14Ac_Rep1,ChIP


title: Met_Bcat_TREAT_BL_181


title: ChAT-GFP negative liver B cells, 4 hours post-PHX, rep 4

description:
Library name: 25_S66
Column name in raw_RSEM_counts.txt: X25_S66

value: PHX


title: RFP+YFP+Tregs_mice2

city: Beijing

first: Ting

last: Wang

address: No.1 Beichen West Road, Chaoyang District, Beijing, China

institute: Institute of Microbiology, Chinese Academy of Sciences

data_processing:
Raw data (raw reads) of fastq format were firstly processed through in-house perl script
Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5.
The mapped reads of each sample were assembled by StringTie (v1.3.3b) (Mihaela Pertea.et al. 2015) in a reference-based approach
featureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene
Differential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq2 R package (1.16.1)
Assembly: mm10
Supplementary files format and content: tab-delimited Excel files include RPKM values for each Sample

source_name: Tregs

extract_protocol:
Lymphocytes were isolated from the spleen and lymph nodes of 6- to 8-week-old Foxp3RFP-Cre.RosaYFPMll1fl/fl male mice and enriched for CD4+ T cells using magnetic beads. RFP+YFP+ and RFP+YFP- Tregs (3 x 105) were sorted to a typical purity of > 95 for RNA sequencing.RNA was extracted using the RNeasy MicroRNA Kit (QIAGEN 74004).
Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample

value: Tregs

tag: cell subtype

value: RFP+YFP+


title: Met_Bcat_CTRL_BL_180


title: ChAT-GFP positive liver B cells, 4 hours post-PHX, rep 5

description:
Library name: 3_S45
Column name in raw_RSEM_counts.txt: X3_S45

value: GFP-Pos


title: ChAT-GFP positive liver B cells, 4 hours post-PHX, rep 3

description:
Library name: 24_S65
Column name in raw_RSEM_counts.txt: X24_S65


title: RFP+YFP-Tregs_mice2

value: RFP+YFP-

value: WT


title: Met_Bcat_TREAT_BL_184


title: Arabidopsis, Mock,H3, 12DAG, Rep2

description: Library name: 12DAG_Mock_H3_Rep2,ChIP

value: 12DAG seedling


title: RFP+YFP+Tregs_mice1


title: Arabidopsis, Mock,H3, 12DAG, Rep1

description: Library name: 12DAG_Mock_H3_Rep1,ChIP


title: Met_Bcat_CTRL_BL_179

description:
Met_Bcat_CTRL_BL_179
179 and 178 are the same sample.


title: Arabidopsis, h2a.z-5,HTA67, 12DAG, Rep2

description:
Library name: 12DAG_h2a.z-5_HTA67_Rep2,ChIP
h2a.z-5 triple mutant line (carries extra adenosine in HTA8 after base 241 causing a frameshift and a premature stop codon after base 318, SALK_054814 and, SALK_017235)

value: CD771，杭州景杰


title: ChAT-GFP positive liver B cells, 4 hours post-PHX, rep 2

description:
Library name: 23_S64
Column name in raw_RSEM_counts.txt: X23_S64


title: RFP+YFP-Tregs_mice1


title: Met_Bcat_CTRL_BL_177


title: ChAT-GFP negative liver B cells, 4 hours post-PHX, rep 3

description:
Library name: 22_S63
Column name in raw_RSEM_counts.txt: X22_S63

value: GFP-Neg


title: H3K4me3_RFP+YFP+ Tregs_mice2

data_processing:
Run fastqc and multiqc
Run trimmomatic
Run hisat2
Run samtools
Use bamCoverage to generate bigwig file, run macs2 peak calling
Assembly: mm10
Supplementary files format and content: bigwig
Library strategy: Cut&Tag

extract_protocol:
Lymphocytes were isolated from the spleen and lymph nodes of 6- to 8-week-old Foxp3-Cre.RosaYFP and Foxp3-Cre.RosaYFPMll1fl/fl male mice or Foxp3RFP-Cre.RosaYFPMll1fl/fl male mice and enriched for CD4+ T cells using magnetic beads. YFP+Tregs or RFP+YFP+ and RFP+YFP- Tregs (1 x 105) were sorted to a typical purity of > 95 for CUT&TAG sequencing.CUT&Tag libraries were generated with the Hyperactive In-Situ ChIP Library Prep Kit for Illumina Kit (Vazyme Biotech, TD901).
Briefly, 50,000 cells for each CUT&Tag library were incubated with 100 μL balanced concanavalin A-coated magnetic beads at RT for 10 min. The bead-bound cells were resuspended with 50 μL of antibody buffer. Then added 1 ug anti-H3K4me3 antibody (Abcam, ab8580) to the bead-bound cells. After 2 h incubation at RT, the primary antibody was discarded carefully and 0.5 μg goat anti-rabbit IgG (Vazyme, Ab206-10-AA) diluted with 50μL Dig-wash buffer was added to the cells at RT for 1 h. Afterward, samples were washed gently with 800 μL Dig-wash buffer, and 2 μL pG–Tn5 together with 100 μL Dig-300 buffer was added to the samples. After 1 h incubation at RT, samples were washed gently with 800 μL Dig-300 buffer. 10 μL tagmentation buffer together with 40 μL Dig-300 buffer was then added to each sample and the samples were incubated at 37°c for 1 h. The interactions were quenched by adding 10 μL 0.5 M EDTA, 3 μL 10% SDS, and 2.5 μL 20 mg/mL Proteinase K and incubating the samples at 50°c for 1 h. Phenol–chloroform and ethanol precipitation, as well as PCR and size selection, were performed as described in Hi-C library construction. All CUT&Tag libraries were sequenced on Illumina Nova Seq 6000 platform at PE150 mode subsequently.

tag: chip antibody


title: Arabidopsis, Mock,H3K9K14Ac, 12DAG, Rep2

description: Library name: 12DAG_Mock_H3K9K14Ac_Rep2,ChIP


title: ChAT-GFP positive liver B cells, sham surgery, rep 5

description:
Library name: 21_S62
Column name in raw_RSEM_counts.txt: X21_S62

value: Sham


title: Arabidopsis, h2a.z-5,HTA67, 12DAG, Rep1

description:
Library name: 12DAG_h2a.z-5_HTA67_Rep1,ChIP
h2a.z-5 triple mutant line (carries extra adenosine in HTA8 after base 241 causing a frameshift and a premature stop codon after base 318, SALK_054814 and, SALK_017235)


title: ChAT-GFP negative liver B cells, sham surgery, rep 5

description:
Library name: 20_S61
Column name in raw_RSEM_counts.txt: X20_S61


title: Aged

city: San Francisco

first: Juliana

last: Sucharov Costa

state: CA

address: 505 Parnassus Ave

department: Anatomy

institute: UCSF

data_processing:
The raw base sequence calls were demultiplexed into sample-specific cDNA files with bcl2fastq/mkfastq sample sheet using Cell Ranger 7.1 (10x Genomics). Raw FASTQ files were processed and aligned to mm10 using the Cell Ranger software package (10x Genomics) for the RNA expression matrix, including introns in analysis for single nucleus analysis. Outputs were integrated with Cell Ranger Aggr (10x Genomics) to normalize for sequencing depth. For CD8+ adoptive transfer sequencing, a total of 52,221 cells were sequenced at a depth of 37,013 reads per cell and approximately a 68% sequencing saturation of 34.1%.
Downstream analysis and data visualization was performed using Seurat. Data were processed to remove doublets and unwanted sources of variation by removing cells with more than 3,500 and fewer than 300 genes per cell and regressing on number of UMIs. Genes expressed in fewer than three nuclei were filtered out. Nuclei with a percentage of mitochondrial genes of higher than 0.1% were removed. Final nuclei counts of 14,589 (Young CD8), 10,510 (Aged CD8), 24,463 (Ptx CD8) were used for gene expression analysis. The matrices of data were log-normalized in a sparse data matrix and PCA was applied to reduce dimensionality. The first 30 PCA components were used to cluster cells by Louvain clustering implemented in Seurat while UMAP plots were independently generated to aid in 2D representation of multidimensional data independent of the clustering.
Assembly: mm10
Supplementary files format and content: Filtered tab-separated values and matrix files

molecule: nuclear RNA

source_name: Hippocampus

extract_protocol:
Mice were anesthetized with 87.5 mg per kg ketamine and 12.5 mg per kg xylazine and transcardially perfused with ice-cold phosphate-buffered saline. To process the brains, the whole brain was sectioned in half along the sagittal plane. The hippocampus from one hemisphere was subdissected and flash-frozen. For adoptive transfer with PTx, neuronal nuclei were isolated based on the demonstrated protocol by 10x Genomics with modifications and performed on nuclei isolated from three mice per group. Briefly, flash-frozen dissected hippocampi, three samples per group, were dounce homogenized (Wheaton, Cat# 357538) in 500μL of NP40 lysis buffer (10mM Tris-HCl (pH = 7.4), 10mM NaCl, 3mM MgCl2, 0.1% Nonidet P40 Substitute), 1mM DTT, 1x RNAse Inhibitor, Nuclease-free H2O) with 20 strokes of the loose pestle and 25 strokes of the tight pestle. 500 μL of NP40 lysis buffer was added and samples incubated for 7 minutes on ice. Samples were filtered through a 40 μm filter and centrifuged at 500 RCF for 5 min at 4°C. Samples were resuspended in 1ml of Wash Buffer (PBS, 1%BSA, 1xRNAse Inhibitor) and incubated for 5 min on ice. Samples were centrifuged at 500 RCF for 5 min at 4°C, supernatant was removed, and samples were resuspended in 400 uL of Wash Buffer with 1:10,000 dilution of Hoescht 33342 and incubated for five minutes before filtering through a 35μm FACS tube filter and sorting on a BD FACSAria II. Nuclei were sorted on a BD FACSAria Fusion with a 70 μm nozzle and with a flow rate of 1–2.5. Nuclei were first gated by forward and side scatter, then gated for doublets with height and width. Nuclei that were Hoechst+ were sorted and samples were combined per group.
Samples were delivered to the UCSF-IHG Genomics Core for analysis with the 10x Genomics Chromium Single Cell Expression Solution 3′ kit with Feature Barcode Technology (v.3.1). The Genomics Core prepared cells for 10x Genomics Chromium single-cell capture. 30,000 nuclei were loaded per sample. cDNA libraries were prepared according to the standard 10x Genomics protocols. The final library pool was sequenced on the NovaSeq X 10B system at the UCSF CAT Core.

treatment_protocol: To investigate functionally how aged CD8+ T exerted their pro-aging effects on the young hippocampus, we pre-treated aged CD8+ T cells with pertussis toxin (PTx), a known inhibitor of Gi/o protein signaling, to block migration into lymph nodes following adoptive transfer and performed single nucleus RNA sequencing (snRNAseq) on the hippocampi from each group. Specifically, B6.SJL-Ptprca Pepcb/BoyJ (4 months) mice were treated i.p. with 200 μm of InVivoMAb anti-mouse CD8a in 200 μL PBS day -11 and -7 to deplete resident CD8+ T cells. CD8+ T cells were isolated from single cell suspensions of spleenocytes and PBMCs from young (3-4 months) or aged (22-28 months) C57BL/6J mice using the CD8a+ T cell Isolation Kit (Miltenyi Biotech, 130-104-075) according to manufacturer instructions. For pertussis toxin treatment (PTx) (Thermo Fisher Scientific, PHZ1174), cells were incubated at a concertation of 2e6 cells/mL in T Cell Media (RPMI + 10% FBS + 1% p/s, NAA, L-glut, NaPyr + 50 μM BME) with IL-2 (1:2000, stock 1000 U/μL, Thermo Fisher, 212-12) and 0.5 μg/mL PTx or vehicle control. Cells were incubated for 24 hours, then were washed three times with saline before resuspension at concentrations of 3e5 cells/100 μL saline (first injection, d0) or 3e6 cells/100 μL saline (second injection, d11) and injection via tail vein. Each mouse was injected i.v. with 100uL of cells in saline (3e5-3e6 cells/injection) and sacrificed 24 days after first transfer.

value: Hippocampus

value: B6.SJL-Ptprca Pepcb/BoyJ

value: Adoptive transfer of vehicle treated aged CD8+ T cells


title: PTx

value: Adoptive transfer of PTx treated aged CD8+ T cells


title: H3K4me3_RFP+YFP- Tregs_mice2


title: Arabidopsis, Columbia,HTA67, 12DAG, Rep1

description: Library name: 12DAG_Columbia_HTA67_Rep1,ChIP


title: Arabidopsis, Columbia,HTA67, 12DAG, Rep2

description: Library name: 12DAG_Columbia_HTA67_Rep2,ChIP


title: H3K4me3_RFP+YFP+ Tregs_mice1


title: ChAT-GFP positive liver B cells, sham surgery, rep 4

description:
Library name: 1_S43
Column name in raw_RSEM_counts.txt: X1_S43

value: Liver

value: Bcell


title: A_iso

data_processing:
The raw base sequence calls were demultiplexed into sample-specific cDNA files with bcl2fastq/mkfastq sample sheet using Cell Ranger 7.1 (10x Genomics). Raw FASTQ files were processed and aligned to mm10 using the Cell Ranger software package (10x Genomics) for the RNA expression matrix. Outputs were integrated with Cell Ranger Aggr (10x Genomics) to normalize for sequencing depth. For parabiosis sequencing, a total of 59,846 cells were sequenced at a depth of 30,977 reads per cell and approximately a 68% sequencing saturation.
Downstream analysis and data visualization was performed using Seurat. Data were processed to remove doublets and unwanted sources of variation by removing cells with more than 3,500 and fewer than 300 genes per cell and regressing on number of UMIs. Genes expressed in fewer than three cells or nuclei were filtered out. Cells with a percentage of mitochondrial genes of higher than 10% were removed. Final cell counts of 8341 (Young Iso), 8418 (Young Het 45.1), 9981 (Young Het 45.2), 5694 (Aged Het 45.1), 8480 (Aged Het 45.2), and 7364 (Aged Iso) were used for gene expression analysis. The matrices of data were log-normalized in a sparse data matrix and PCA was applied to reduce dimensionality. The first 20 PCA components were used to cluster cells by Louvain clustering implemented in Seurat while UMAP plots were independently generated to aid in 2D representation of multidimensional data independent of the clustering.
Assembly: mm10
Supplementary files format and content: Filtered tab-separated values and matrix files

source_name: Spleenocytes from Aged Isochronic control

extract_protocol:
Mice were anesthetized with 87.5 mg per kg ketamine and 12.5 mg per kg xylazine and transcardially perfused with ice-cold phosphate-buffered saline. For collection of splenocytes, spleens were removed, mechanically dissociated with a syringe plunger over a 70 μm cell strainer and washed with 10 ml of MACS. Cells were centrifuged and RBC lysis was performed (155 mM NH4Cl, 1 mM KHCO3 and 0.1 mM EDTA) with 2 ml for 5 minutes room temperature (RT). Subsequently, cells were washed, spun 500 RCF for 5 minutes, and resuspended in MACS. ScRNAseq analysis was performed on splenocytes isolated from four mice per group. Splenocytes were isolated as described above, blocked (2.4G2), then labeled with CD45.1 (A20, 1:200) and C45.2 (104, 1:200) antibodies in MACS for 30 minutes on ice. Samples were washed, then stained with a live/dead marker (Hoechst 33342, 1:10,000) before filtering through a 35 μm FACS tube filter sorting on a BD FACSAria Fusion with a 70 μm nozzle and with a flow rate of 1–2.5. Cells were first gated by forward and side scatter, then gated for doublets with height and width. 40,000 cells per sample that were Hoechst- and CD45.1+ or CD45.2+ were collected for sequencing analysis and pooled into 6 groups (CD45.1+ young isochronic, CD45.1+ young heterochronic, CD45.2+ young heterochronic, CD45.1 + aged heterochronic, CD45.2+ aged heterochronic, and CD45.2+ aged isochronic).
Samples were delivered to the UCSF-IHG Genomics Core for analysis with the 10x Genomics Chromium Single Cell Expression Solution 3′ kit with Feature Barcode Technology (v.3.1). The Genomics Core prepared cells for 10x Genomics Chromium single-cell capture. 25,000 cells were loaded per sample. cDNA libraries were prepared according to the standard 10x Genomics protocols. The final library pool was sequenced to a depth of 30,000 cDNA reads per cell and 3,000 ADT reads per cell on the NovaSeq X 10B system at the UCSF CAT Core.

treatment_protocol: To assess changes in peripheral CD8+ T cell trafficking and transcriptional responses to a young and aging systemic milieu using heterochronic parabiosis in combination with single cell RNA sequencing (scRNAseq). For parabiosis surgery, pairs of C57BL/6J (22 months, aged) or B6.SJL-Ptprca Pepcb/BoyJ (3 months, young) mice were anaesthetized and prepared for surgery. Mirror-image incisions at the left and right flanks were made through the skin, and shorter incisions were made through the abdominal wall. The peritoneal openings of the adjacent parabionts were sutured together. Elbow and knee joints from each parabiont were sutured together, and the skin of each mouse was stapled (9-mm Autoclip, Clay Adams) to the skin of the adjacent parabiont. Each mouse was injected subcutaneously with enrofloxacin (Bayer) antibiotic, buprenorphine (Butler Schein), and carprofen (Rimadyl) as directed for pain and monitored during recovery. For overall health and maintenance behavior, recovery characteristics of paired weights and grooming were analyzed at various times after surgery. Parabionts were sacrificed 4 weeks after surgery.

value: Spleen

value: Spleenocytes

value: Parabiosis


title: H3K4me3_RFP+YFP- Tregs_mice1

value: Tregs


title: A_het_45.2

source_name: Spleenocytes from Aged heterochronic mouse, CD45.2+ (Aged cells)


title: A_het_45.1

source_name: Spleenocytes from Aged heterochronic mouse, CD45.1+ (Young cells)


title: Arabidopsis, hda9-1,H3K27Ac, 12DAG, Rep1

description: Library name: 12DAG_hda9-1_H3K27Ac_Rep1,ChIP

value: 39135，Active Motif


title: Arabidopsis, hda9-1,H3K27Ac, 12DAG, Rep2

description: Library name: 12DAG_hda9-1_H3K27Ac_Rep2,ChIP


title: ChAT-GFP positive liver B cells, 4 hours post-PHX, rep 1

description:
Library name: 17_S58
Column name in raw_RSEM_counts.txt: X17_S58

value: Male

value: B lymphocyte

value: PHX

value: GFP-Pos


title: Y_het_45.2

source_name: Spleenocytes from Young heterochronic mouse, CD45.2+ (Aged cells)


title: ChAT-GFP negative liver B cells, sham surgery, rep 4

description:
Library name: 18_S59
Column name in raw_RSEM_counts.txt: X18_S59

value: GFP-Neg


title: H3K4me3_YFP+_KO1

value: YFP+


title: ChAT-GFP negative liver B cells, 4 hours post-PHX, rep 2

description:
Library name: 16_S57
Column name in raw_RSEM_counts.txt: X16_S57


title: H3K4me3_YFP+_WT2

value: WT


title: Arabidopsis, Columbia,H3K27Ac, 12DAG, Rep2

description: Library name: 12DAG_Columbia_H3K27Ac_Rep2,ChIP

value: 12DAG seedling


title: Y_het_45.1

source_name: Spleenocytes from Young heterochronic mouse, CD45.1+ (Young cells)

value: B6.SJL-Ptprca Pepcb/BoyJ


title: Young

value: Hippocampus

value: Adoptive transfer of vehicle treated young CD8+ T cells


title: ChAT-GFP negative liver B cells, sham surgery, rep 3

description:
Library name: 15_S56
Column name in raw_RSEM_counts.txt: X15_S56

value: Liver

value: Sham


title: Arabidopsis, Columbia,H3K27Ac, 12DAG, Rep1

description: Library name: 12DAG_Columbia_H3K27Ac_Rep1,ChIP


title: H3K4me3_YFP+_WT1


title: Arabidopsis, hda6-1,H3K18Ac, 12DAG, Rep2

description: Library name: 12DAG_hda6-1_H3K18Ac_Rep2,ChIP


title: Arabidopsis, TSA,12DAG, Rep5

description: Library name: 12DAG_TSA_Group2_Rep5

tag: treatment/control

value: Treatment


title: Arabidopsis, hda6-1,H3K18Ac, 12DAG, Rep1

description: Library name: 12DAG_hda6-1_H3K18Ac_Rep1,ChIP


title: PRV+CUR 3

city: chongqin

first: luqiu

last: feng

address: 160 Xueyuan Road, Rongchang District, Chongqing

department: College of Veterinary Medicine

institute: Southwest university

data_processing:
HiSeqTM2500/4000 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to mm8 whole genome using bowtie v0.12.2 with parameters -q -p 4 -e 100 -y -a -m 10 --best --strata
In order to make samples with different gene lengths, different experiments and different sequencing data volumes comparable, people introduced the concept of FPKM. FPKM (Fragments per kilobase of Exon Model per million mapped reads) is the number of Fragments per thousand base length from a gene in each million reads. FPKM takes into account the effects of sequencing depth and gene length on Reads count, and is the most commonly used method to measure gene expression level at present.HTSeq software was used to analyze the gene expression level of each sample, and the model was UNION. The number of genes and the expression level of individual genes at different expression levels were counted in the results file. In general, the FPKM value of 0.1 or 1 is used as the threshold of gene expression.
There was first normalization of the original Read count, which mainly corrected the sequencing depth. Then, the hypothesis testing probability (P-value) is calculated by statistical model. Finally, multiple hypothesis testing correction (BH) was performed to obtain the FDR value (error detection rate, PADJ is the common form, padJ is used to represent FDR below).
Assembly: mm9
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample …
Supplementary files format and content: Matrix table with raw gene counts for every gene and every sample

source_name: brain microglial cell

growth_protocol: Bv2 was stored in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics in humid air at 37℃ and 5% CO2

extract_protocol:
Bv2 cells with different treatments were taken out and quickly frozen on dry ice. RNA was collected using Trizol reagent.  The NEBNext® Ultra™ RNA Library Prep Kit for Illumina® and 1 ug total RNA were used to construct the sequencing Library.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: brain microglial cell

tag: cell line

value: microglial cell


title: Y_iso

source_name: Spleenocytes from Young Isochronic control

value: Spleen

value: Spleenocytes

value: Parabiosis


title: ChAT-GFP positive liver B cells, sham surgery, rep 2

description:
Library name: 14_S55
Column name in raw_RSEM_counts.txt: X14_S55

value: Male

value: B lymphocyte


title: PRV+CUR 2


title: ChAT-GFP negative liver B cells, sham surgery, rep 2

description:
Library name: 13_S54
Column name in raw_RSEM_counts.txt: X13_S54

value: GFP-Neg


title: Arabidopsis, TSA,12DAG, Rep4

description: Library name: 12DAG_TSA_Group2_Rep4


title: ChAT-GFP positive liver B cells, sham surgery, rep 3

description:
Library name: 19_S60
Column name in raw_RSEM_counts.txt: X19_S60

value: GFP-Pos


title: Arabidopsis, Columbia,H2A.X, 12DAG, Rep1

description: Library name: 12DAG_Columbia_H2A.X_Rep1,ChIP


title: H3K4me3_YFP+_KO2

value: Tregs

value: YFP+


title: PRV+CUR 1

value: WT


title: ChAT-GFP negative liver B cells, sham surgery, rep 1

description:
Library name: 12_S53
Column name in raw_RSEM_counts.txt: X12_S53


title: Arabidopsis, TSA,12DAG, Rep3

description: Library name: 12DAG_TSA_Group2_Rep3

value: 12DAG seedling


title: PRV 3


title: ChAT-GFP positive liver B cells, sham surgery, rep 1

description:
Library name: 11_S52
Column name in raw_RSEM_counts.txt: X11_S52


title: PRV 2


title: Arabidopsis, Columbia,H3K18Ac, 12DAG, Rep1

description: Library name: 12DAG_Columbia_H3K18Ac_Rep1,ChIP


title: Arabidopsis, TSA,12DAG, Rep2

description: Library name: 12DAG_TSA_Group2_Rep2


title: Arabidopsis, hda9-1,H3K9K14Ac, 12DAG, Rep2

description: Library name: 12DAG_hda9-1_H3K9K14Ac_Rep2,ChIP


title: ChAT-GFP negative liver B cells, 4 hours post-PHX, rep 1

description:
Library name: 10_S51
Column name in raw_RSEM_counts.txt: X10_S51

value: PHX


title: Arabidopsis, TSA,12DAG, Rep1

description: Library name: 12DAG_TSA_Group2_Rep1


title: Control 3


title: Arabidopsis, Mock,12DAG, Rep5

description: Library name: 12DAG_Mock_Group2_Rep5

value: Control


title: BL_152 LNP-CTNNB1 R Phenotype

value: liver

value: BL_152 LNP-CTNNB1 R Phenotype


title: Arabidopsis, hda9-1,H3K9K14Ac, 12DAG, Rep1

description: Library name: 12DAG_hda9-1_H3K9K14Ac_Rep1,ChIP


title: Arabidopsis, hda6-1,H3K9K14Ac, 12DAG, Rep2

description: Library name: 12DAG_hda6-1_H3K9K14Ac_Rep2,ChIP


title: BL_147 LNP-CTNNB1 NR phenotype

value: BL_147 LNP-CTNNB1 NR phenotype


title: Arabidopsis, Mock,12DAG, Rep4

description: Library name: 12DAG_Mock_Group2_Rep4


title: Control 2

value: brain microglial cell

value: microglial cell


title: Arabidopsis, Mock,12DAG, Rep3

description: Library name: 12DAG_Mock_Group2_Rep3


title: Arabidopsis, Columbia,H3K9K14Ac, 12DAG, Rep2

description: Library name: 12DAG_Columbia_H3K9K14Ac_Rep2,ChIP


title: Control 1


title: Arabidopsis, Columbia,H3K18Ac, 12DAG, Rep2

description: Library name: 12DAG_Columbia_H3K18Ac_Rep2,ChIP


title: BL_146 LNP-CTNNB1 NR phenotype

value: BL_146 LNP-CTNNB1 NR phenotype


title: Arabidopsis, Mock,12DAG, Rep2

description: Library name: 12DAG_Mock_Group2_Rep2


title: BL_131 LNP-CTRL

value: BL_131 LNP-CTRL


title: Arabidopsis, Columbia,H3K9K14Ac, 12DAG, Rep1

description: Library name: 12DAG_Columbia_H3K9K14Ac_Rep1,ChIP


title: MDS C22

description: 10x Genomics

data_processing:
Reads from single-cell RNA-Seq experiments were aligned to the human genome (version hg38) and quantified using the count function in CellRanger v4.0.0 from 10x Genomics and using gene models from Ensembl as the reference transcriptome.
Assembly: hg38
Supplementary files format and content: hdf5 files of cell barcodes and read counts produced by CellRanger Count

source_name: HSPCs

extract_protocol:
Freshly collected cells were passage through columns for cell removal to achieve a viability of >90%. Cells were resuspended in PBS/0/04% BSA (PBS without EDTA and Mg). 100 µl of single cell suspension at concentration of 700-1200 cells/µl was submitted to the sequencing service.
Library was performed according to the manufacturers instruction (3' scRNAseq v3.1 10x Chromium)

value: HSPCs

value: MDS low risk

value: no treatment


title: Arabidopsis, Mock,12DAG, Rep1

description: Library name: 12DAG_Mock_Group2_Rep1


title: Arabidopsis, hda9-1,H2A.Z, 12DAG, Rep2

description: Library name: 12DAG_hda9-1_H2A.Z_Rep2,ChIP


title: PRV 1


title: Pooled 3 tumor_control + 3 tumor_treat samples, FB

library_source: other

data_processing:
Demultiplexing was performed by 10X Genomics cellranger multi function, using the hashtag configuration file
For each of the six samples, the aligned BAM file were converted into FASTQ by bamtofastq
For each sample, the 10X Genomics cellranger count was applied to each FASTQ data
Assembly: mm10
Supplementary files format and content: barcodes.tsv.gz: TSV file
Supplementary files format and content: features.tsv.gz: TSV file
Supplementary files format and content: matrix.mtx.gz: MTX file

molecule: protein

extract_protocol:
Hepatocytes isolated and purified from whole liver perfusions along with the enriched nonparenchymal cell fraction (mostly lymphocytes/monocytes) were used for tandem sc-RNA-Seq
RNA libraries were prepared by 10X Genomics for single-cell perfusion, and hashtag strategy, followed by sequencing using standard Illumina protocols.

tag: library type

value: FB


title: Arabidopsis,hda9-1 h2a.z-5 mutant,12DAG, Rep3

description: Library name: 12DAG_hda9-1 h2a.z-5_Group1_Rep3

value: hda9-1 h2a.z-5 mutant

value: Treatment


title: MDS C22.7

description: 10x Genomics

value: MDS High risk


title: Arabidopsis,hda9-1 h2a.z-5 mutant,12DAG, Rep2

description: Library name: 12DAG_hda9-1 h2a.z-5_Group1_Rep2

value: 12DAG seedling


title: Arabidopsis, hda6-1,H2A.Z, 12DAG, Rep2

description: Library name: 12DAG_hda6-1_H2A.Z_Rep2,ChIP


title: Arabidopsis, hda6-1,H3K9K14Ac, 12DAG, Rep1

description: Library name: 12DAG_hda6-1_H3K9K14Ac_Rep1,ChIP


title: Arabidopsis, hda9-1,H2A.Z, 12DAG, Rep1

description: Library name: 12DAG_hda9-1_H2A.Z_Rep1,ChIP


title: BU3.10

description: 10x Genomics

value: wild type


title: tumor_treat

data_processing:
10X Cell Ranger was used to aligned the reads to mouse reference genome
Assembly: mm10
Supplementary files format and content: barcodes.tsv.gz: TSV file
Supplementary files format and content: features.tsv.gz: TSV file
Supplementary files format and content: matrix.mtx.gz: MTX file

extract_protocol:
Hepatocytes isolated and purified from whole liver perfusions along with the enriched nonparenchymal cell fraction (mostly lymphocytes/monocytes) were used for tandem sc-RNA-Seq
RNA libraries were prepared by 10X Genomics followed by sequencing using standard Illumina protocols.

value: Treatment with LNP-CTRL/LNP-CTNNB1


title: tumor_control

value: liver

value: Control


title: BU3.10 erythroid

description: 10x Genomics

source_name: Erythroid

value: Erythroid


title: Arabidopsis, hda6-1,H2A.Z, 12DAG, Rep1

description: Library name: 12DAG_hda6-1_H2A.Z_Rep1,ChIP


title: Arabidopsis,hda9-1 mutant,12DAG, Rep3

description: Library name: 12DAG_hda9-1_Group1_Rep3


title: Arabidopsis, Columbia,H2A.Z, 12DAG, Rep2

description: Library name: 12DAG_Columbia_H2A.Z_Rep2,ChIP


title: S1PR1-deficient SVZ-NSCs, ApoM+HDL, rep2

city: Daegu

first: Hee Kyung

last: Jin

address: 80 Daehakro, Buk-gu

country: South Korea

institute: Kyungpook National University

data_processing:
Illumina bcl2fastq2-v2.20.0 software used for basecalling.
Trimmomatic program is used to remove adapter sequences and bases with base quality lower than three from the ends. Also using sliding window method, bases of reads that does not qualify for window size 4, and mean quality 15 are trimmed. Afterwards, reads with length shorter than 36bp are dropped to produce trimmed data.
Sequenced reads were mapped to GRCm38 (mm10) whole genome by using HISAT2 version 2.1.0 with parameter -p 20 -dta --rna-strandness RF --novel-splicesite-outfile -x -1 -2
Fragment Per Kilobase of exon per Megabase of library size (FPKM), Transcripts Per Million (TPM) were calculated by using StringTie version 2.1.3b, with parameter --rf -p 20 -l -o -G -A -C -e
Assembly: mm10
Supplementary files format and content: tab-delimited text files include FPKM values, TPM, read counts for each Sample

source_name: SVZ-NSCs

extract_protocol:
RNA was harvested using RNeasy® Mini Kit (Qiagen), 0.5ug of Total RNA was used fot construction of sequencing libraries.
RNA library for RNA-seq were prepared using TruSeq Stranded Total RNA with Ribo-Zero H/M/R_Gold library prep kit following manufacturer's protocols

value: SVZ-NSCs

value: S1pr1 deletion


title: Pooled 3 tumor_control + 3 tumor_treat samples, GEX

value: mRNA


title: S1PR1-deficient SVZ-NSCs, ApoM+HDL, rep1


title: pooled mRNA-derived cDNA and ADT-derived cDNA

city: Boston

first: Johannes

last: Wedel

state: MA

address: 300 Longwood Ave

institute: Boston Children's Hospital

description:
3' mRNA library: read1 file contains cell barcode and UMI; read2 file contains transcript,
Antibody-derived oligonucleotide library: read1 file contains cell barcode and UMI; read2 file contains Antibody Derived Tag reads

data_processing:
Raw BCL files were converted to FASTQ, tsv and mtx files using the Cell Ranger pipeline (version 3.1.0, 10x Genomics, Pleasanton, CA) and imported into Seurat (version 3.2.0) in the R Statistical Computing Environment (version 4.0.0).
Assembly: GRCh38
Supplementary files format and content: 10x Genomics output files: filtered_barcodes.tsv.gz, filtered_features.tsv.gz, filtered_matrix.mtx.gz

source_name: CD4+ T cells

extract_protocol:
mouseCD45neg humanCD3pos humanCD4pos live cells were FACS-sorted 7 days post-transfer. Purified cells were stained with DNAoligo-conjugated anti-NRP2 antibodies and subsequently loaded onto a 10x controller (10x Genomics).
Libraries were prepared using Chromium Single Cell 3ʹ v3 protocol (10x Genomics). Cellular mRNA and antibody-derived oligos were reverse-transcribed and indexed with a shared cellular barcode. Indexed cDNAs were then pooled and amplified by PCR according to the Chromium Single Cell 3’ v3 protocol (10x Genomics) and with specific primers amplifying the antibody-derived tags.

treatment_protocol: SCID-beige mice were humanized by intravenous transfer of 5x10^7 PBMC.

value: CD4+ T cells

value: mRNA + ADT

tag: antibodies/tags

value: Anti-NRP2 (clone MM03; barcode:TGACCAGTTCCGCAT), Anti-NRP2 (clone 2v2; barcode:CGGTATATCAACAGA)


title: Arabidopsis,hda9-1 h2a.z-5 mutant,12DAG, Rep1

description: Library name: 12DAG_hda9-1 h2a.z-5_Group1_Rep1


title: Arabidopsis, Columbia(hda9-1),H2A.Z, 12DAG, Rep1

description: Library name: 12DAG_Columbia(hda9-1)_H2A.Z_Rep1,ChIP


title: S1PR1-deficient SVZ-NSCs, ApoM-HDL, rep2


title: Arabidopsis,hda9-1 mutant,12DAG, Rep2

description: Library name: 12DAG_hda9-1_Group1_Rep2


title: Arabidopsis,hda9-1 mutant,12DAG, Rep1

description: Library name: 12DAG_hda9-1_Group1_Rep1


title: MDS C22 erythroid

description: 10x Genomics

value: MDS low risk

value: no treatment


title: Arabidopsis, Columbia(hda6-1),H2A.Z, 12DAG, Rep1

description: Library name: 12DAG_Columbia(hda6-1)_H2A.Z_Rep1,ChIP


title: S1PR1-deficient SVZ-NSCs, ApoM-HDL, rep1


title: B2-5

type: RNA

hyb_protocol: Protocal provided and performed by resolve biosciences (https://your.resolve-biosciences.com/), with reference paper PMID: 35021063.

data_processing:
Gene count per cell were identified by QuPath
csv:  Gene count per cell (FP or PF followed with a number are internal control probes.)
tiff: indicates the nucleus location
txt: shows the gene expression at a certain location

source_name: liver

extract_protocol: Frozen livers were sectioned and sent on dry ice to resolve bioscience for further processing


title: Arabidopsis,hda6-1 h2a.z-5 mutant,12DAG, Rep3

description: Library name: 12DAG_hda6-1 h2a.z-5_Group1_Rep3

value: hda6-1 h2a.z-5 mutant


title: Arabidopsis,hda6-1 h2a.z-5 mutant,12DAG, Rep2

description: Library name: 12DAG_hda6-1 h2a.z-5_Group1_Rep2

value: Treatment


title: Control SVZ-NSCs, ApoM+HDL, rep2

value: WT


title: B2-4

scan_protocol: Protocal provided and performed by resolve biosciences (https://your.resolve-biosciences.com/), with reference paper PMID: 35021063.


title: B2-3


title: Control SVZ-NSCs, ApoM+HDL, rep1


title: Arabidopsis,hda6-1 mutant,12DAG, Rep3

description: Library name: 12DAG_hda6-1_Group1_Rep3

value: 12DAG seedling


title: B2-2


title: Control SVZ-NSCs, ApoM-HDL, rep1


title: Arabidopsis,hda6-1 mutant,12DAG, Rep2

description: Library name: 12DAG_hda6-1_Group1_Rep2


title: Control SVZ-NSCs, ApoM-HDL, rep2


title: B1-6

value: LNP-CTRL


title: CD8

data_processing:
The raw base sequence calls were demultiplexed into sample-specific cDNA files with bcl2fastq/mkfastq sample sheet using Cell Ranger 7.1 (10x Genomics). Raw FASTQ files were processed and aligned to mm10 using the Cell Ranger software package (10x Genomics) for the RNA expression matrix, including introns in analysis for single nucleus analysis. For CD8+ adoptive transfer sequencing, outputs were integrated with Cell Ranger Aggr (10x Genomics) to normalize for sequencing depth. A total of 52,221 cells were sequenced at a depth of 37,013 reads per cell and approximately a 68% sequencing saturation of 34.1%. For CD8+ depletion sequencing, 15,237-16,194 cells were sequenced at a sequencing depth of 23,213 reads per cell and 37.6% sequencing saturation (IgG2b) or a sequencing depth of 32,910 reads per cell and 47.4% sequencing saturation (CD8a). Ambient RNA was removed with the SoupX package and data from the four samples was integrated using Seurat (2000 variable features, 1:20 dimensions).
Downstream analysis and data visualization was performed using Seurat. Data were processed to remove doublets and unwanted sources of variation by removing cells with more than 3,500 and fewer than 300 genes per cell and regressing on number of UMIs. Genes expressed in fewer than three nuclei were filtered out. Nuclei with a percentage of mitochondrial genes of higher than 0.1% were removed. Final nuclei counts of 14,589 (Young CD8), 10,510 (Aged CD8), 24,463 (Ptx CD8), 12,079 (IgG2b), and 12,062 (CD8a) were used for gene expression analysis. The matrices of data were log-normalized in a sparse data matrix and PCA was applied to reduce dimensionality. The first 20 (depletion) or 30 (adoptive transfer) PCA components were used to cluster cells by Louvain clustering implemented in Seurat while UMAP plots were independently generated to aid in 2D representation of multidimensional data independent of the clustering.
Assembly: mm10
Supplementary files format and content: Filtered and raw  tab-separated values and matrix files

extract_protocol:
Mice were anesthetized with 87.5 mg per kg ketamine and 12.5 mg per kg xylazine and transcardially perfused with ice-cold phosphate-buffered saline. To process the brains, the whole brain was sectioned in half along the sagittal plane. The hippocampus from one hemisphere was subdissected and flash-frozen. For adoptive transfer with PTx, neuronal nuclei were isolated based on the demonstrated protocol by 10x Genomics with modifications and performed on nuclei isolated from three mice per group. Briefly, flash-frozen dissected hippocampi, three samples per group, were dounce homogenized (Wheaton, Cat# 357538) in 500μL of NP40 lysis buffer (10mM Tris-HCl (pH = 7.4), 10mM NaCl, 3mM MgCl2, 0.1% Nonidet P40 Substitute), 1mM DTT, 1x RNAse Inhibitor, Nuclease-free H2O) with 20 strokes of the loose pestle and 25 strokes of the tight pestle. 500 μL of NP40 lysis buffer was added and samples incubated for 7 minutes on ice. Samples were filtered through a 40 μm filter and centrifuged at 500 RCF for 5 min at 4°C. Samples were resuspended in 1ml of Wash Buffer (PBS, 1%BSA, 1xRNAse Inhibitor) and incubated for 5 min on ice. Samples were centrifuged at 500 RCF for 5 min at 4°C, supernatant was removed, and samples were resuspended in 400 uL of Wash Buffer with 1:10,000 dilution of Hoescht 33342 and incubated for five minutes before filtering through a 35μm FACS tube filter and sorting on a BD FACSAria II. Nuclei were sorted on a BD FACSAria Fusion with a 70 μm nozzle and with a flow rate of 1–2.5. Nuclei were first gated by forward and side scatter, then gated for doublets with height and width. Nuclei that were Hoechst+ were sorted and samples were combined per group.
Samples were delivered to the UCSF-IHG Genomics Core for analysis with the 10x Genomics Chromium Single Cell Expression Solution 3′ kit with Feature Barcode Technology (v.3.1). The Genomics Core prepared cells for 10x Genomics Chromium single-cell capture. 30,000 nuclei were loaded per sample. cDNA libraries were prepared according to the standard 10x Genomics protocols. The final library pool was sequenced on the NovaSeq 6000 S2 system (CD8+ depletion) or the NovaSeq X 10B system (CD8+ adoptive transfer) at the UCSF CAT Core.

treatment_protocol: To investigate functionally how aged CD8+ T exerted their pro-aging effects on the young hippocampus, we pre-treated aged CD8+ T cells with pertussis toxin (PTx), a known inhibitor of Gi/o protein signaling, to block migration into lymph nodes following adoptive transfer and performed single nucleus RNA sequencing (snRNAseq) on the hippocampi from each group. Specifically, B6.SJL-Ptprca Pepcb/BoyJ (4 months) mice were treated i.p. with 200 μm of InVivoMAb anti-mouse CD8a in 200 μL PBS day -11 and -7 to deplete resident CD8+ T cells. CD8+ T cells were isolated from single cell suspensions of spleenocytes and PBMCs from young (3-4 months) or aged (22-28 months) C57BL/6J mice using the CD8a+ T cell Isolation Kit (Miltenyi Biotech, 130-104-075) according to manufacturer instructions. For pertussis toxin treatment (PTx) (Thermo Fisher Scientific, PHZ1174), cells were incubated at a concertation of 2e6 cells/mL in T Cell Media (RPMI + 10% FBS + 1% p/s, NAA, L-glut, NaPyr + 50 μM BME) with IL-2 (1:2000, stock 1000 U/μL, Thermo Fisher, 212-12) and 0.5 μg/mL PTx or vehicle control. Cells were incubated for 24 hours, then were washed three times with saline before resuspension at concentrations of 3e5 cells/100 μL saline (first injection, d0) or 3e6 cells/100 μL saline (second injection, d11) and injection via tail vein. Each mouse was injected i.v. with 100uL of cells in saline (3e5-3e6 cells/injection) and sacrificed 24 days after first transfer. To selectively target aged peripheral CD8+ T cells late in life, aged (22-24 months) mice were treated i.p. with 200 μm of InVivoMAb anti-mouse CD8a (2.43, BioXCell, BE0061) or InVivoMAb rat IgG2b isotype control (LTF-2, BioXCell, BE0090) in 200 μL PBS every 4 days until time of sacrifice. Depletion of circulating CD8+ T cells was confirmed with flow cytometry.

value: Hippocampus

tag: age

value: Aged

value: CD8a Depletion


title: Arabidopsis,hda6-1 mutant,12DAG, Rep1

description: Library name: 12DAG_hda6-1_Group1_Rep1


title: IgG

value: IgG2b Isotype Control


title: B1-5

value: liver


title: Arabidopsis,h2a.z-5 triple mutant line,12DAG, Rep2

description:
Library name: 12DAG_h2a.z-5_Group1_Rep2
h2a.z-5 triple mutant line (carries extra adenosine in HTA8 after base 241 causing a frameshift and a premature stop codon after base 318, SALK_054814 and, SALK_017235)

value: h2a.z-5 triple mutant line


title: B1-4


title: Z0317AB0082-R_Col64

city: Cheongju-si

first: Eun-Jeong

last: YOON

address: Osongsaengmyeong-ro

institute: KNIH

data_processing:
RNA library was prepared with 2 µg total RNA using TruSeq RNA Library Prep Kit (Illumina) and its quality was controlled using TapeStation HS D5000 Screening Tape (Agilent Technologies) and LightCycler qPCR. The sequencing data was 2 Gb per sample. Using the read-trimmed raw reads from Illumina HiSeq with a minimum length of 50 bp, Bowtie 2 (ver. 2.3.5.) was employed for mapping and the value of fragments per kilobase of transcript per million mapped reads was used to normalize sequencing depth and transcript length. Differentially expressed gene analysis was conducted using the DEGseq package of R.
Assembly: Genome was builed based on the whole genome sequence by PacBio reads, which was generated for separate experiment for the study
Supplementary files format and content: tab-delimited text file includes raw counts for each sample
Supplementary files format and content: tab-delimited text file include Fragments Per Kilobase of transcript per Million values for each sample and the

organism: Acinetobacter baumannii

source_name: Bacterial cell

growth_protocol: Isolates were grown in Mueller Hinton broth

extract_protocol:
Total RNA was extracted from cultures in the exponential growth phase in MH broth using the Qiagen RNeasy Mini Kit.
RNA library was prepared with 2 µg total RNA using TruSeq RNA Library Prep Kit (Illumina) and its quality was controlled using TapeStation HS D5000 Screening Tape (Agilent Technologies) and LightCycler qPCR.

treatment_protocol: The parental A. baumannii Z0317AB0082 and its isogenic mutant Z0317AB0082-R were induced for 30 min with either 2 µg/ml for both isolates or 64 µg/ml colistin for the isogenic mutant Z0317AB0082-R

value: Bacterial cell

value: isogenic mutant resistant to colistin

value: inhibitory concentration of colistin


title: LLCshIKKA

city: Munich

first: Georgios

middle: T

last: Stathopoulos

state: Bayern

address: Max-Lebsche-Platz

department: Comprehensive Pneumology Center

country: Germany

institute: Helmholz Zentrum Munich

description: Normalization and summarization performed with RMA algorithm

hyb_protocol: Arrays were prepared according to Affymetrix GeneChip™ Hybridization, Wash, and Stain Kit

scan_protocol: Scanner 3000 7G

data_processing: Expression console software and Transcriptome console software 3.0 from Affymetrix

source_name: lung cancer cell lines; lentiviral transduction of shIKKA

label_protocol: Arrays were prepared according to Affymetrix specifications with GeneChip™ WT Pico Reagent Kit

growth_protocol: cells were cultured in minimum supplemented medium (DMEM+10% FBS + 1% PEN/STREP)

extract_protocol: Total RNA from the cells was extracted with the Trizol protocol followed by Qiagen RNeasy Mini Kit

treatment_protocol: Subculturing of plasmid or shRNA expressing cell lines is done in DMEM + 10% FBS + 4-6 µM puromycin

value: lung cancer cell line

value: LLC

tag: genotype/variation

value: IKKA knockdown


title: BMDM-MC38

source_name: bone-marrow macrophages from mouse inoculated with MC38 cell line

value: bone-marrow macrophages

value: mouse inoculated with MC38 cell line


title: BMDM-Tomato-Lys

source_name: bone-marrow macrophages from Tomato-Lys mouse

value: mouse expressing Tomato-Lys


title: MC38wt

source_name: colon carcinoma cell line

value: colon carcinoma cell line

value: wildtype


title: LLCwt

source_name: lung cancer cell lines


title: Arabidopsis,h2a.z-5 triple mutant line,12DAG, Rep1

description:
Library name: 12DAG_h2a.z-5_Group1_Rep1
h2a.z-5 triple mutant line (carries extra adenosine in HTA8 after base 241 causing a frameshift and a premature stop codon after base 318, SALK_054814 and, SALK_017235)

data_processing:
(1)Filter:ChIP-seq reads were trimmed using the fastp tool (version 0.23.4)  with a quality score threshold of >20 for the final base.Reads shorter than 35 bp after trimming were discarded.(2)Alignment:The clean reads were aligned to the TAIR 10 genome (https://www.arabidopsis.org/) using Bowtie2 and sorted and indexed using the samtools suite (version 1.17).(3)Remove: PCR duplicates were removed using Picard (version 3.0.0). (4)Callpeak:Peaks were identified using the “callpeak” function of MACS2 (version 2.2.6,). (5)Coverage tracks were calculated: normalizing using RPGC (effective genome size 119482912) –bin size 10, normalization method using RPGC reads per genome coverage), creating bigwig files.(6)Plot:The metagene results were drawn using deepTools(version 3.5.2).
Assembly: TAIR 10
Supplementary files format and content: BigWig files were generated using the Deeptools  (version 3.5.2).The BigWig files for making plot were  assessed by merging results from 'bigwigcompare' of two independent biological replicates, employing the 'mean' operation.

source_name: 12DAG seedling

extract_protocol:
1)Crosslink protein-DNA interactions with formaldehyde; 2) Fragment chromatin via sonication or enzymatic digestion; 3) Immunoprecipitate target protein-DNA complexes using specific antibodies; 4) Reverse crosslinking and purify DNA, followed by library construction for sequencing .
ChIP-Seq libraries were prepared using Ovation® Ultralow System V2 Library Preparation Kit (PART NO. 0344, 0344NB).

tag: tissue

value: 12DAG seedling

value: Treatment


title: Z0317AB0082-R_Col2

tag: treatment

value: subinhibitory concentration of colistin


title: Mtz, 5dpf

city: Solna

first: Rodrigo

middle: A.

last: Morales Castro

state: Stockholms län

address: Karolinska Universitetssjukhuset, CMM L8:03

country: Sweden

institute: Karolinska Institutet

data_processing:
Demultiplexing of raw Illumina sequencing files, barcoding processing, gene counting and aggregation were performed using the CellRanger v3.0 software
Assembly: Ensembl GRCz11.105
Supplementary files format and content: Tab-separated values files and matrix files

organism: Danio rerio

source_name: Dissected larval intestines

extract_protocol:
Larval intestines were manually dissected within an interval of 2 hours and collected in sterile HBSS-BSA 0.5%. Single-cell preparations were done by incubating dissected intestines in dissaggregation buffer (TrypLE supplemented with 0.5mg/mL of Dnase I) for 30min at 28°C, with extensive pipetting every 10 minutes. Single cell preparations were washed with HBSS-BSA 0.5%, filtered with a 70µm membrane, and stained with DAPI 0.2 mg/mL to exclude dead cells. Live intestinal cells were FACS-sorted using a Sony SH800S cell sorter.
Library was prepared according to manufacturer's instructions (10x Genomics).

value: Dissected larval intestines

value: Mix of intestinal cells

value: AB

value: Metronidazole


title: Arabidopsis,hda6-1 h2a.z-5 mutant,12DAG, Rep1

description: Library name: 12DAG_hda6-1 h2a.z-5_Group1_Rep1

value: hda6-1 h2a.z-5 mutant


title: BMDM-LLC

source_name: bone-marrow macrophages from mouse inoculated with LLC cell line

value: mouse inoculated with LLC cell line


title: B2-1

value: liver


title: B1-3

value: LNP-CTRL


title: Z0317AB0082-R_NT

value: Bacterial cell

value: isogenic mutant resistant to colistin

value: control


title: Control, 5dpf

value: Control


title: Arabidopsis, Columbia,12DAG, Rep3

description: Library name: 12DAG_Columbia_Group1_Rep3


title: B1-2


title: BMDM-IKK1-Lys

source_name: bone-marrow macrophages from Tomato-Lys_IKK1 mouse

value: bone-marrow macrophages

value: conditionally deleted for IKKA


title: Z0317AB0082_Col2

value: wild type

value: inhibitory concentration of colistin


title: B16F10wt

source_name: naturally derived skin tumor and development of immortal cell line

value: skin tumor

value: wildtype


title: Mtz, ablation 4dpf

value: Dissected larval intestines

value: Mix of intestinal cells


title: Arabidopsis, Columbia,12DAG, Rep2

description: Library name: 12DAG_Columbia_Group1_Rep2

value: 12DAG seedling


title: Control, ablation 4dpf

value: AB


title: Arabidopsis,h2a.z-5 triple mutant line,12DAG, Rep3

description:
Library name: 12DAG_h2a.z-5_Group1_Rep3
h2a.z-5 triple mutant line (carries extra adenosine in HTA8 after base 241 causing a frameshift and a premature stop codon after base 318, SALK_054814 and, SALK_017235)

value: h2a.z-5 triple mutant line

value: Treatment


title: BMDM-IKK2-Lys

source_name: bone-marrow macrophages from Tomato-Lys_IKK2 mouse

value: conditionally deleted for IKKB


title: Z0317AB0082_NT

value: Bacterial cell


title: Arabidopsis, Columbia,12DAG, Rep1

description: Library name: 12DAG_Columbia_Group1_Rep1


title: E14.5 epidermis, intermediate cells, biol rep3

status: Public on May 01 2025

city: Durham

first: Alexandra

last: Prado Mantilla

state: NC

address: 307 Research Dr, Durham, NC 27710

department: Cell Biology

institute: Duke University

description:
Library name: IC_E14_3
E14_5I_3

data_processing:
Reads were trimmed and aligned to the mouse reference genome mm10 (GRCm38) using STAR (v2.5). Gene-level quantification was performed using HTSeq (v0.6.1) and expression values were calculated as FPKM. Differential gene expression analysis was performed using the DESeq2 R package (v1.6.3), with p-values adjusted for false discovery using the Benjamini-Hochberg method.
Assembly: mm10 (GRCm38)
Supplementary files format and content: Excel spreadsheet includes FPKM values for all samples

source_name: E14.5 embryonic back skin epidermis

extract_protocol:
K10-rtTA;TRE-H2B-GFP;K14-RFP pregnant dams were all fed with doxycycline chow at E12.5, and then sacrificed at either E14.5 or E16.5. Embryos were checked under a dissecting microscope for GFP and RFP expression before use, and their tails were taken to confirm genotypes. E16.5 and E14.5 back skins were cut off and treated with 1.4 U/ml dispase II (Roche, 4942078001) in HBSS at room temperature for 1 hour, then peeled off the epidermis from the dermis. The epidermis of E14.5 back skin was not peeled off due to its low thickness, but was still treated with dispase II to maintain the same conditions as the E16.5 samples. Both E16.5 epidermis and E14.5 skin samples were digested in 1:1 trypsin (GIBCO, 25200-056) with versene (GIBCO, 15040-066) at 37°C and rotated for 20 minutes. Samples were then mixed 1:1 with FACS buffer (HBSS with 2.5% FBS and 10 μg/ml DNAaseI) and centrifuged at low speed. The cell pellet was diluted into FACS buffer with propidium iodide solution (Sigma, P4864, to exclude dead cells), and filtered using sterile Celltrics 30 μm filters (Sysmex, 04-004-2326). For E16.5 samples, we excluded granular cells by using  Ulex europaeus agglutinin I lectin (UEA1), which binds to the more superficial cells. For this, we used biotinylated UEA1 (Vector Laboratories, B-1065) and detected them with APC-streptavidin (BioLegend, 405207).  The E14.5 intermediate cells (K10-GFP+;K14-RFP-;APC-UEA1-), E16.5 spinous cells (K10-GFP+;K14-RFP-;APC-UEA1-), and basal cells at E14.5 and E16.5 (K10-GFP-;K14-RFP+) cells were FACS sorted separately. RNA was extracted using a QIAGEN RNAeasy Mini kit (QIAGEN, 74104) following the manufacturer’s protocols, with DNA digested using RNase-Free DNase (QIAGEN, 79254). Biological replicates, three independent RNA samples from each cell population, were collected and sent for sequencing and analysis by Novogene.
NEB library: Total RNA was extracted and mRNA was purified using poly-T oligo-attached magnetic beads. The mRNA was randomly fragmented using fragmentation buffer, and first strand cDNA synthesis was performed using random hexamer primers and M-MuLV Reverse Transcriptase. Second strand cDNA was synthesized using DNA Polymerase I and RNase H. The resulting double-stranded cDNA was purified using AMPure XP beads, and end-repair was performed to generate blunt ends. After adenylation of 3′ ends, NEBNext adaptors with a hairpin loop structure were ligated. Size selection was performed to enrich for fragments of ~150–200 bp. Libraries were amplified by PCR and purified using AMPure XP beads.

treatment_protocol: Doxycycline chow since E12.5

value: E14.5 embryonic back skin epidermis

value: suprabasal cells

value: keratinocytes

tag: batch


title: E16.5 epidermis, spinous cells, biol rep3

description:
Library name: Spinous_E16_3
E16_5Sp3

source_name: E16.5 embryonic back skin epidermis

value: E16.5 embryonic back skin epidermis

tag: cell type


title: E14.5 epidermis, intermediate cells, biol rep1

description:
Library name: IC_E14_1
E14_5I_1

tag: genotype


title: E16.5 epidermis, spinous cells, biol rep2

description:
Library name: Spinous_E16_2
E16_5Sp2


title: E16.5 epidermis, spinous cells, biol rep1

description:
Library name: Spinous_E16_1
E16_5Sp1


title: E14.5 epidermis, intermediate cells, biol rep2

description:
Library name: IC_E14_2
E14_5I_2


title: AngII+E1_4

city: Wuhan

first: MENGMENG

address: Wuhan University

institute: Wuhan University

description:
Library name: Sample14
AngII+E1_4

data_processing:
CLC Genomics Workbenchv 11.0.1
Assembly: mm10
Supplementary files format and content: tab-delimited text file includes raw counts for each Sample

source_name: aorta

extract_protocol:
RNA was harvested using Rneasy mini plus kit (Qiagen). 1.3 ug of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA seg were prepared using SMARTER mRNA-Seq LibraryPrep Kit following manufacturer's protocols.

value: aorta

value: WT

value: Ang II (angiotensin II) infusion + E1 ip


title: B1-1

value: liver

value: LNP-CTRL


title: E16.5 epidermis, basal cells, biol rep3

description:
Library name: Basal_E16_3
E16_5Bc3

value: basal cells


title: AngII_2

description:
Library name: Sample7
AngII_2

value: Ang II (angiotensin II) infusion


title: E16.5 epidermis, basal cells, biol rep2

description:
Library name: Basal_E16_2
E16_5Bc2


title: Sham_4

description:
Library name: Sample4
Sham_4

value: Sham


title: AngII_1

description:
Library name: Sample6
AngII_1


title: E14.5 epidermis, basal cells, biol rep2

description:
Library name: Basal_E14_2
E14_5Bc2


title: E14.5 epidermis, basal cells, biol rep1

description:
Library name: Basal_E14_1
E14_5Bc1


title: E16.5 epidermis, basal cells, biol rep1

description:
Library name: Basal_E16_1
E16_5Bc1


title: AngII+E1_2

description:
Library name: Sample12
AngII+E1_2


title: AngII+E1_3

description:
Library name: Sample13
AngII+E1_3


title: AngII+E1_1

description:
Library name: Sample11
AngII+E1_1


title: AngII_4

description:
Library name: Sample9
AngII_4


title: Sham_3

description:
Library name: Sample3
Sham_3


title: AngII_5

description:
Library name: Sample10
AngII_5


title: AngII_3

description:
Library name: Sample8
AngII_3


title: Sham_1

description:
Library name: Sample1
Sham_1


title: Sham_2

description:
Library name: Sample2
Sham_2


title: KO13

city: Tianjin

first: shuaibing

last: hou

address: Nanjing road 288

institute: State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases

data_processing:
Illumina Casava1.7 software used for basecalling
Sequenced reads were trimmer for adaptor sequence,and masked for low-complexity or low-quality sequence,then mapped to mm8 whole genome using bowtie v0.12.0 with parameters
Reads Per Kilobase of exon per Megabase of library size(RPKM)were calculated using a protocol from Chepelev et al,Nucleic Acids Research,2009.In short,exons from all isofroms.
Assembly: mm8
Supplementary files format and content: tab-delimited text files include RPKM values for each Sample

source_name: Bone marrow

growth_protocol: The GFP+ leukemia cells of bone marrow were sorted from VC Phf6,RE9a and WT Phf6,RE9a mouse models

extract_protocol:
1x107 Cells was resuspend using Trizol reagent.Illumina TruSeq RNA Sample Prep Kit was used with 1μg of total RNA for the construction
RNA libraries were prepared for sequencing using standard Illumina protocols.

value: Bone marrow

value: leukemia cells

value: GFP+ Cells

value: phf6 specific konckout in hematopoietic system


title: AngII+E1_5

description:
Library name: Sample15
AngII+E1_5


title: WT2

value: Phf6 WT


title: KO12


title: WT3


title: E14.5 epidermis, basal cells, biol rep3

description:
Library name: Basal_E14_3
E14_5Bc3

value: E14.5 embryonic back skin epidermis


title: Rag2NE1KO2

city: Chicago

first: Amy

last: Kenter

state: IL

address: 835 S Wolcott

institute: UIC

data_processing:
Raw reads were trimmed to remove Truseq adapters and bases from the 3’ end with quality scores less than 20 using cutadapt (Martin, 2011); trimmed reads shorter than 40bp were discarded. Trimmed reads were aligned to the reference genome using STAR (Dobin et al., 2013). ENSEMBL genes were quantified using FeatureCounts (Liao et al., 2014).
Strand-specific enrichment tracks (bigWig) were generated by first converting the bam files to bed using bedtools bamtobed, reversing the orientation of R2 reads to match that of R1. Separate bedgraph files for + and - strands were created using bedtools genomecov using the “-strand” flag, and counts were normalized to reads per billion bases in the library. Bedgraph files were converted to bigWig using UCSC tool bedGraphToBigWig.
Assembly: mm10
Supplementary files format and content: bigwig files were generated after converting the mm10 files to mm9

source_name: BM Pro-B cells

growth_protocol: Pro-B cells were isolated from Rag deficient murine bone marrow using anti-CD19 magnetic microbeads (MACS, Miltenyi) according to the manufacturer’s instructions. B cells cultured 1x10^6 cells/ml in RPMI, glutamine (4 mM), penicillin (200 U/ml)-streptomycin (200 mg/ml), b-mercaptoenthanol (50 mM), supplemented with FCS (10% vol/vol.).   Rag deficient pro-B cells that were cultured with IL7 and expanded in ex vivo culture for 5 days.

extract_protocol: Total RNA was prepared from Rag2-/- and Rag2-/-NE1-/- pro-B cells. RNA was extracted using TRIzol (Life Technologies) and genomic DNA (gDNA) was eliminated using the genomic DNA wipeout buffer in the QuantiTect Reverse Transcription Kit (Qiagen). RNA was purified using with the RNeasy Kit (Qiagen), ribosomal RNA was eliminated using the Ribo-Zero Magnetic Gold Kit (Illumina), processed with the NEBNext Ultra Directional RNA Library Prep Kit (Illumina) and sequenced on the Illumina HiSeq system.

tag: strain

value: BM derived Rag2NE1KO pro-B cells

value: ex vivo IL7 expansion  5 days


title: Rag2NE1KO3


title: Rag2KO3

value: BM derived Rag2KO pro-B cells


title: Rag2NE1KO1


title: Sham_5

description:
Library name: Sample5
Sham_5

value: aorta


title: Rag2KO2


title: Rag2KO1


title: STIM1 Kozak library cells replicate 3

city: Cleveland

first: Kenneth

last: Matreyek

state: OH

address: 2103 Cornell Rd

department: Pathology

institute: Case Western Reserve University

description:
Library name: ML_STIM1_r3_cells
Supp_table_2_mixed_library_infection.csv

data_processing:
For short-read sequencing generated by the Illumina NextSeq, the forward and reverse reads were paired using PEAR (Zhang et al. 2014). Kozak sequences were counted from the paired reads using Enrich2 (Rubin et al. 2017), and filtered to exclude reads possessing nucleotides with quality scores less than 20. For longer-read sequencing generated by the Illumina MiSeq, generated through submission to the Amplicon-EZ service offered by Genewiz / Azenta, the reads were paired as above, except the paired reads were subsequently processed with a custom Python script “Extract_Kozak.py”, which identified true Kozak library reads from template molecule reads, and for each Kozak read, extracted the Kozak sequence found in that read. All subsequent analyses of the sequencing outputs were performed in R version 4.3.1 (2023-06-16). An R markdown file with code to recreate the analysis (Kozak.Rmd), along with the relevant data files and the various python scripts for the initial processing of FASTQ files, can be found in the MatreyekLab Github repository under “MatreyekLab/Kozak”. A table of the fastq files generated and analyzed for this work is included as Supplementary Table 4. The Kozak sort-seq scores were calculated similarly to previous work (Matreyek et al, 2018, 2021), with some minor changes. For samples sequenced to sufficient read depth, a density-plot of sequence counts normally yields a bimodal distribution, with one peak stemming from a log-normal distribution of counts with a median of 10 or greater, largely reflecting sequences of physical molecules amplified and sequenced from the sample, and a separate peak with a count of 1, largely reflecting sequences derived from sequencing error. We harnessed this property to computationally remove erroneous sequence counts. For each bin, we identified the local minima between these two peaks, and removed sequence counts below this sample-specific threshold value, to yield a dataset of filtered counts. For each sequenced sample, the filtered count of each variant was divided by the sum of counts recorded in that sample to obtain variant-specific frequencies (Fv,bin). For replicates 1 and 2, which both included separately sequenced amplification “technical” replicates derived from the same sorted genomic DNA, the technical replicate frequencies were averaged to yield an average frequency for each variant, which was used as the Fv,bin value. These frequencies were used to calculate a weighted average for each variant for each replicate (Wv,rep), using the following formula.  Wv,rep =(Fv,bin1 0) + (Fv,bin2 (1/3)) + (Fv,bin3 (2/3)) + (Fv,bin4 1)Fv,bin1 + Fv,bin2 + Fv,bin3 + Fv,bin4   Thus, all weighted average values ranged from a value of 0 to 1. Pairwise comparisons of variant-specific weighted averages are shown in Supplementary Fig 2. Once the weighted average values were calculated for each variant for each replicate, they were log10 transformed, averaged, and the base-10 exponential calculated to yield the geometric mean weighted average value. Correspondingly, the standard deviation of the log10 transformed values were calculated and used to calculate the upper and lower 95% confidence intervals, which were later transformed as a base-10 exponential to accompany the geometric mean weighted averages.  To calibrate these weighted averages to MFI equivalent values, we generated cells recombined with ACE2-miRFP670 constructs encoding 16 different Kozak sequences, which were measured for miRFP670 fluorescence intensity using analytical flow cytometry. A linear model was first generated to establish the conversion factor enabling scaling of our weighted average scores to MFI-equivalent values. While accurate for values between 1% and 100% of the consensus Kozak, the linear model performed poorly for extremely high or low values, with the minimum and maximum scaled values being 0.0003 and 3.89; both of these values were outside of the range physically observed with this assay. We thus used logistic regression to approximate the linear model, but furthermore bound all scaled values between 0.004 and 1.34. These resulting values are referred to as the calibrated scores.  Enrichment values for infection experiments were initially filtered similarly to the sort-seq scores, wherein we identified the local minima between the two peaks observed in a density plot representation of sequence counts, and removed sequence counts below his sample-specific threshold value, to yield a dataset of filtered counts. The counts for each variant were divided by the total counts for that sequence sample, to yield sample-specific variant frequencies. For infection samples that included separately sequenced amplification “technical” replicates derived from the same sorted genomic DNA, the geometric means of the technical replicate frequencies were used. Each hygromycin-selected infected sample had a corresponding unselected sample. For each pair, we divided variant-specific frequencies following selection by those prior to selection, obtaining an enrichment ratio. The STIM1 survival scores were calculated similarly, with the exception that the ratio was the quotient of each Kozak variant frequency recombined into cells divided by the frequency of that variant in the sequenced plasmid library.
Assembly: Custom synthetic amplicon
Supplementary files format and content: csv file with calibrated sort-seq scores and infection scores
Supplementary files format and content: csv file with ACE2 variant-specific infection scores

organism: synthetic construct

source_name: HEK 293T

extract_protocol:
Genomic DNA was isolated from each sample using the JetFlex™ Genomic DNA purification kit (Thermo Fisher, A30700).
ACE2 amplicon generation and high throughput sequencing A section of DNA spanning the recombination junction, and including the Kozak sequence preceding the ACE2 open reading frame, was amplified from the genomic DNA extracts using PCR. For each 50 µL PCR reaction, the following components were used: 2 µg genomic DNA (quantified by nanodrop), 0.25 µM  forward primer containing i7 index barcode and 0.25 µM  reverse primer containing i5 index barcode, and 25 µL of Phusion plus high fidelity PCR mix (Thermo Fisher, F548L). A total of five PCR reactions were performed per condition, which each tube initially incubated at 95 °C for 3 minutes, followed by 25 cycles of 95 °C for 15 seconds, 60 °C for 15 seconds, 72 °C for 30 seconds, followed by a final 72 °C incubation for 1 minute. PCR products were mixed with 6x DNA loading dye (NEB, B7024S) and separated on 1.5% agarose TAE gel. Bands corresponding to the expected 293 bp amplicon size were excised and extracted using the Freeze and Squeeze kit according to the manufacturer’s instructions (Bio-Rad, 7326166). Eluted PCR products were purified using a Zymo clean and concentrator kit (Zymo Research, D4003). Amplicon concentrations were measured using Qubit dsDNA BR assay kit (Life technologies, Q32853). Amplicons from all the experimental conditions were pooled in equimolar ratios and sequenced on an Illumina NextSeq550 with a 75-cycle high output kit. Oligonucleotide sequences for PCR and custom Illumina primers for sequencing are listed in Supplementary Table 3.

value: HEK 293T

value: G542Ac3 landing pad


title: STIM1 Kozak library cells replicate 2

description:
Library name: ML_STIM1_r2_cells
Supp_table_2_mixed_library_infection.csv


title: STIM1 Kozak library plasmid

description:
Library name: ML_STIM1_plasmid
Supp_table_2_mixed_library_infection.csv

molecule: other


title: STIM1 Kozak library cells replicate 1

description:
Library name: ML_STIM1_r1_cells
Supp_table_2_mixed_library_infection.csv


title: mat2a_wt_rep_2

city: Vilnius

first: Kotryna

last: Kvederaviciute

address: Sauletekio av. 7

department: Life Sciences Center

country: Lithuania

institute: Vilnius University

data_processing:
Raw data quality was evaluated using FASTQC. Reads were trimmed using cutadapt (v3.4) and in-house script.
Cleaned reads were mapped to the reference genome (mm10) using bwa (v0.7.17). Samtools (v1.9) were used to perform all manipulations with SAM/BAM files. samclip was used to remove soft-clipped data.
Each reads was assigned to a specific CG target and coverage tables were generated using an in-house R (v.4.3.0) script.
Assembly: mm10
Supplementary files format and content: Tab delimited file with CpG coverages.
Library strategy: Dnmt-TOP-seq

source_name: Mouse embryonic stem E14TG2a cell line

growth_protocol: For the experiment cells were plated in 24-well plates at a density of 2,5 x 104cells per well and grown for 48h in DMEM media supplemented with mLIF

extract_protocol:
Embryonic stem cells were resuspended in 100μL PBS and and 100 μ Lysis buffer (0.2M Tris-HCl, 0.25M NaCl, 0.025M EDTA, 0.5% SDS, pH 8) were added, next the samples were supplemented with 6 U Proteinase K  and incubated for 40min at 56°C. Further, samples were treated with0,02 U RNaseA  for 10min at room temperature, genomic DNA was isolated using Genomic DNA Clean & Concentrator-10 Kit.
Libraries were constructed according to the TOP-seq (Tethered Oligonucleotide-Primed sequencing) protocol (Stasevskij et al., 2017). Briefly, 900 ng of sonicated genomic DNA were end-filled, a 3’-dA mononucleotide extension was added and DNA was ligated to adapters. Ligated DNA was then subjected to Cu(I)-catalyzed cycloaddition reaction in which the alkyne-bearing oligodeoxyribonucleotide with biotin was attached to the azide moiety of the modified DNA. Biotinylated DNA was enriched with streptavidin-coupled magnetic beads and subsequently used in a TOP DNA priming reaction from the attached priming oligodeoxyribonucleotide. The products were amplified and subjected to Ion Proton sequencing.

treatment_protocol: Cells were fed with 2 mM S-(6-azidohex-2-yn-1-yl)-L-homocysteine in the DMEM media with 200 µM methionine an incubated with methionine analog for 24 h

value: Mouse embryonic stem E14TG2a cell line

value: Dnmt1 wt, Mat2A wt


title: mat2a_I117A_rep2

value: Engineered Dnmt1 and Mat2A  double knock-in


title: mat2a_I117A_rep1


title: mat2a_wt_rep_3


title: mat2a_I117A_rep3


title: LU481_PDX_p5-1

city: South San Francisco

first: Hin Ching

last: Lo

address: 1000 Gateway Blvd

institute: AbbVie Inc.

description:
Library name: LU481_PDX_p5-1
Column name in RNAseq_logTPM.csv: LU481_PDX_p5.1

data_processing:
FastQC (version v0.11.8) was applied to check the quality of raw reads.
Trimmomatic (version v0.38) was applied to cut adaptors and trim low-quality bases with default setting.
STAR Aligner version 2.7.1a was used to align the reads.
The featureCounts (version 1.6.0)/HTSeq was used to count mapped reads for genomic features such as genes, exons, promoter, gene bodies, genomic bins and chromosomal locations.
The StringTie version 2.0.4 was used to assemble the RNA-Seq alignments into potential transcripts.
Assembly: GRCh38
Supplementary files format and content: csv file with log2TPM+1 for each sample

source_name: Human PDX tumor cells

growth_protocol: Patient-derived xenograft (PDX) tumor cells were implanted and propagated in female NOD/SCID mice (5 to 10 weeks of age; Charles River Laboratories). For spheroid samples, ultra-low attachment plates containing PDX-derived spheroids were maintained in culture in 37°C and 5% CO2 in humidified conditions.

extract_protocol:
RNA was extracted from tumor spheroids and matched PDX tumors (purified for human cells) using the RNeasy mini Kit (Qiagen).
RNA library preparation (NEBNext Ultra RNA Library Prep Kit for Illumina, NEBNext Poly(A) mRNA Magnetic Isolation Module) and sequencing (Illumina NovaSeq S4, 150 nucleotide paired-end reads) were performed by Admera Health (South Plainfield, NJ).

treatment_protocol: No treatment was applied on samples.

value: Human PDX tumor cells

value: WT


title: LU481_sp_p5-3

description:
Library name: LU481_sp_p5-3
Column name in RNAseq_logTPM.csv: LU481_sp_p5.3


title: LU481_PDX_p5-2

description:
Library name: LU481_PDX_p5-2
Column name in RNAseq_logTPM.csv: LU481_PDX_p5.2


title: WT1

value: Bone marrow


title: LU554_sp_p3-3

description:
Library name: LU554_sp_p3-3
Column name in RNAseq_logTPM.csv: LU554_sp_p3.3


title: KO11

value: leukemia cells


title: LU554_PDX_p3-1

description:
Library name: LU554_PDX_p3-1
Column name in RNAseq_logTPM.csv: LU554_PDX_p3.1


title: LU554_sp_p3-1

description:
Library name: LU554_sp_p3-1
Column name in RNAseq_logTPM.csv: LU554_sp_p3.1


title: LU554_sp_p3-2

description:
Library name: LU554_sp_p3-2
Column name in RNAseq_logTPM.csv: LU554_sp_p3.2


title: LU481_sp_p5-1

description:
Library name: LU481_sp_p5-1
Column name in RNAseq_logTPM.csv: LU481_sp_p5.1


title: LU450_sp_p3-3

description:
Library name: LU450_sp_p3-3
Column name in RNAseq_logTPM.csv: LU450_sp_p3.3


title: LU554_PDX_p3-2

description:
Library name: LU554_PDX_p3-2
Column name in RNAseq_logTPM.csv: LU554_PDX_p3.2


title: LU450_sp_p3-2

description:
Library name: LU450_sp_p3-2
Column name in RNAseq_logTPM.csv: LU450_sp_p3.2


title: LU450_sp_p3-1

description:
Library name: LU450_sp_p3-1
Column name in RNAseq_logTPM.csv: LU450_sp_p3.1


title: LU450_PDX_p3-2

description:
Library name: LU450_PDX_p3-2
Column name in RNAseq_logTPM.csv: LU450_PDX_p3.2


title: LU554_sp_p2-3

description:
Library name: LU554_sp_p2-3
Column name in RNAseq_logTPM.csv: LU554_sp_p2.3


title: LU450_PDX_p3-1

description:
Library name: LU450_PDX_p3-1
Column name in RNAseq_logTPM.csv: LU450_PDX_p3.1


title: LU554_sp_p2-2

description:
Library name: LU554_sp_p2-2
Column name in RNAseq_logTPM.csv: LU554_sp_p2.2


title: LU554_sp_p2-1

description:
Library name: LU554_sp_p2-1
Column name in RNAseq_logTPM.csv: LU554_sp_p2.1


title: LU554_sp_p2_nobead-2

description:
Library name: LU554_sp_p2_nobead-2
Column name in RNAseq_logTPM.csv: LU554_sp_p2_nobead.2


title: mat2a_wt_rep_1


title: LU554_sp_p2_nobead-1

description:
Library name: LU554_sp_p2_nobead-1
Column name in RNAseq_logTPM.csv: LU554_sp_p2_nobead.1


title: LU481_sp_p2-2

description:
Library name: LU481_sp_p2-2
Column name in RNAseq_logTPM.csv: LU481_sp_p2.2


title: LU554_PDX_p2-2

description:
Library name: LU554_PDX_p2-2
Column name in RNAseq_logTPM.csv: LU554_PDX_p2.2.y


title: LU554_PDX_p2-1

description:
Library name: LU554_PDX_p2-1
Column name in RNAseq_logTPM.csv: LU554_PDX_p2.1.y


title: LU481_sp_p5-2

description:
Library name: LU481_sp_p5-2
Column name in RNAseq_logTPM.csv: LU481_sp_p5.2


title: LU481_sp_p2-1

description:
Library name: LU481_sp_p2-1
Column name in RNAseq_logTPM.csv: LU481_sp_p2.1


title: LU554_sp_p2_nobead-3

description:
Library name: LU554_sp_p2_nobead-3
Column name in RNAseq_logTPM.csv: LU554_sp_p2_nobead.3


title: LU481_sp_p2_nobead-3

description:
Library name: LU481_sp_p2_nobead-3
Column name in RNAseq_logTPM.csv: LU481_sp_p2_nobead.3


title: LU481_sp_p2-3

description:
Library name: LU481_sp_p2-3
Column name in RNAseq_logTPM.csv: LU481_sp_p2.3


title: LU481_sp_p2_nobead-2

description:
Library name: LU481_sp_p2_nobead-2
Column name in RNAseq_logTPM.csv: LU481_sp_p2_nobead.2


title: LU481_PDX_P4-2

description:
Library name: LU481_PDX_P4-2
Column name in RNAseq_logTPM.csv: LU481_PDX_P4.2


title: LU481_sp_p2_nobead-1

description:
Library name: LU481_sp_p2_nobead-1
Column name in RNAseq_logTPM.csv: LU481_sp_p2_nobead.1


title: LU481_PDX_P4-1

description:
Library name: LU481_PDX_P4-1
Column name in RNAseq_logTPM.csv: LU481_PDX_P4.1


title: LU450_sp_p2-2

description:
Library name: LU450_sp_p2-2
Column name in RNAseq_logTPM.csv: LU450_sp_p2.2


title: LU450_PDX_p2-1

description:
Library name: LU450_PDX_p2-1
Column name in RNAseq_logTPM.csv: LU450_PDX_p2.1


title: LU211_PDX_p8-3

description:
Library name: LU211_PDX_p8-3
Column name in RNAseq_logTPM.csv: LU211_PDX_p8.3


title: LU450_PDX_p2-2

description:
Library name: LU450_PDX_p2-2
Column name in RNAseq_logTPM.csv: LU450_PDX_p2.2


title: LU450_sp_p2-1

description:
Library name: LU450_sp_p2-1
Column name in RNAseq_logTPM.csv: LU450_sp_p2.1


title: LU211_PDX_p8-1

description:
Library name: LU211_PDX_p8-1
Column name in RNAseq_logTPM.csv: LU211_PDX_p8.1


title: LU211_PDX_p8-2

description:
Library name: LU211_PDX_p8-2
Column name in RNAseq_logTPM.csv: LU211_PDX_p8.2


title: LU211_sp_p5-1

description:
Library name: LU211_sp_p5-1
Column name in RNAseq_logTPM.csv: LU211_sp_p5.1


title: LU211_PDX_p5-3

description:
Library name: LU211_PDX_p5-3
Column name in RNAseq_logTPM.csv: LU211_PDX_p5.3


title: LU211_sp_p5-3

description:
Library name: LU211_sp_p5-3
Column name in RNAseq_logTPM.csv: LU211_sp_p5.3


title: LU211_sp_p5-2

description:
Library name: LU211_sp_p5-2
Column name in RNAseq_logTPM.csv: LU211_sp_p5.2


title: LU211_PDX_p5-2

description:
Library name: LU211_PDX_p5-2
Column name in RNAseq_logTPM.csv: LU211_PDX_p5.2


title: LU211_PDX_p5-1

description:
Library name: LU211_PDX_p5-1
Column name in RNAseq_logTPM.csv: LU211_PDX_p5.1


title: LU120_sp_p4_nobead-2

description:
Library name: LU120_sp_p4_nobead-2
Column name in RNAseq_logTPM.csv: LU120_sp_p4_nobead.2


title: LU120_sp_p4_nobead-1

description:
Library name: LU120_sp_p4_nobead-1
Column name in RNAseq_logTPM.csv: LU120_sp_p4_nobead.1


title: LU120_sp_p4-3

description:
Library name: LU120_sp_p4-3
Column name in RNAseq_logTPM.csv: LU120_sp_p4.3


title: LU120_sp_p4-1

description:
Library name: LU120_sp_p4-1
Column name in RNAseq_logTPM.csv: LU120_sp_p4.1


title: LU120_PDX_p4-2

description:
Library name: LU120_PDX_p4-2
Column name in RNAseq_logTPM.csv: LU120_PDX_p4.2


title: LU120_PDX_p4-3

description:
Library name: LU120_PDX_p4-3
Column name in RNAseq_logTPM.csv: LU120_PDX_p4.3


title: LU120_PDX_p4-1

description:
Library name: LU120_PDX_p4-1
Column name in RNAseq_logTPM.csv: LU120_PDX_p4.1


title: LU450_sp_p2-3

description:
Library name: LU450_sp_p2-3
Column name in RNAseq_logTPM.csv: LU450_sp_p2.3


title: HepG2 cells, lytac3, 48 hours

city: Yiwu

first: Huiping

last: Liu

state: Zhejiang

address: Futian Sub-district

institute: Zhejiang University

description: Library name: lytac3

data_processing:
Reads obtained from the sequencing machines includes raw reads containing adapters or low quality bases which will affect the following assembly and analysis. Thus, to get high quality clean reads, reads were further filtered by Cutadapt (https://cutadapt.readthedocs.io/en/stable/,version:cutadapt-1.9). We aligned reads of all samples to the research species reference genome using HISAT2 (https://daehwankimlab.github.io/hisat2/,version:hisat2-2.2.1) package, which initially remove a portion of the reads based on quality information accompanying each read and then maps the reads to the reference genome.
The mapped reads of each sample were assembled using StringTie (http://ccb.jhu.edu/software/stringtie/,version:stringtie-2.1.6) with default parameters. Then, all transcriptomes from all samples were merged to reconstruct a comprehensive transcriptome using gffcompare software(http://ccb.jhu.edu/software/stringtie/gffcompare.shtml,version:gffcompare-0.9.8). After the final transcriptome was generated, StringTie and ballgown (http://www.bioconductor.org/packages/release/bioc/html/ballgown.html) were used to estimate the expression levels of all transcripts and perform expression abundance for mRNAs by calculating FPKM (fragment per kilobase of transcript per million mapped reads) value.
Genes differential expression analysis was performed by DESeq2 software between two different groups (and by edgeR between two samples). The genes with the parameter of false discovery rate (FDR) below 0.05 and absolute fold change ≥ 2 were considered differentially expressed genes. Differentially expressed genes were then subjected to enrichment analysis of GO functions and KEGG pathways.
Assembly: GRCh38
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample

source_name: Liver

extract_protocol:
Total RNA was extracted using Trizol reagent (thermofisher, 15596018) following the manufacturer's procedure. The total RNA quantity and purity were analysis of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA, 5067-1511) , high-quality RNA samples with RIN number > 7.0 were used to construct sequencing library.
mRNA was purified from total RNA (5ug) using Dynabeads Oligo (dT) (Thermo Fisher, CA, USA) with two rounds of purification. Following purification, the mRNA was fragmented into short fragments using divalent cations under elevated temperature (Magnesium RNA Fragmentation Module (NEB, cat.e6150, USA) under 94℃ 5-7min). Then the cleaved RNA fragments were reverse-transcribed to create the cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, cat. 1896649, USA), which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat.m0209, USA), RNase H (NEB, cat.m0297, USA) and dUTP Solution (Thermo Fisher, cat.R0133, USA). An A-base was then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contained a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB, cat.m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min. The average insert size for the final cDNA librarys were 300±50 bp. At last, we performed the 2×150bp paired-end sequencing (PE150) on an Illumina NovaseqTM 6000 following the vendor's recommended protocol.

value: Liver

value: 48 hours


title: HepG2 cells, lytac2, 48 hours

description: Library name: lytac2


title: HepG2 cells, Ordinary medium2, 48 hours

description: Library name: control2


title: RNAseq_501mel shNIPBL_#5 6D rep2

city: New York

first: Elissa

middle: WP

last: Wong

state: NY

address: 1275 York Avenue

department: HOPP

institute: Memorial Sloan Kettering Cancer Center

description: Library name: Sample 6

data_processing:
RNA-seq reads were trimmed and filtered for quality using Trimmomatic (v0.38).
Processed reads were then aligned to hg19 using STAR (v2.7.1a).
For each GENCODE (v19) gene, reads were counted using featureCounts (v1.6.4).
Gene counts were used to perform differential analysis using DESeq2 (v1.22.2).
TPM normalized bigWig tracks were generated using deepTools (v3.1.10 bamCoverage with `--normalizeUsing BPM`) for each replicate. BedGraph files from each replicate, converted from bigWig using bigWigToBedGraph, were combined using bedtools (v2.27.1) unionbedg and scores were averaged for each condition. The averaged bedgraph files were converted to TPM averaged bigWig tracks using bedGraphToBigWig (v4). TPM counts was generated with stringtie (v2.1.1) using BAM files and GENCODE (v19) gtf.
Assembly: hg19
Supplementary files format and content: TPM normalized gene count
Supplementary files format and content: Differential gene expression analysis by DESeq2

source_name: skin

growth_protocol: All cells lines were cultured in media supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. All cells were cultured at 37 ºC in 5% CO2.

extract_protocol:
Cells were lyzed directly on culture dish with TRIzol RNA isolation reagent (Thermo fisher scientific). Total RNA was extracted by thorough mixing with chloroform and isopropanol precipitation of upper aqueous phase.
500 ng of total RNA was used for poly-A selection of RNA and library construction using TruSeq stranded mRNA LT Kit (Illumina, RS-122-2102).
Samples were barcoded and run on a HiSeq 4000 in PE100 to generate 40-50 M reads.

treatment_protocol: Cells that were transduced with lentivirus carrying puromycin-resistant shRNA plasmids were treated with 2 µg/ml puromycin.

value: skin

value: melanoma


title: HepG2 cells, Ordinary medium1, 48 hours

description: Library name: control1


title: RNAseq_501mel shNIPBL_#4 6D rep2

description: Library name: Sample 4


title: HepG2 cells, Ordinary medium3, 48 hours

description: Library name: control3


title: RNAseq_501mel shNIPBL_#5 6D rep1

description: Library name: Sample 5


title: RNAseq_501mel shNIPBL_#4 6D rep1

description: Library name: Sample 3


title: RNAseq_501mel shLuc 6D rep4

description: Library name: Sample 2

value: shLuc


title: LU120_sp_p4_nobead-3

description:
Library name: LU120_sp_p4_nobead-3
Column name in RNAseq_logTPM.csv: LU120_sp_p4_nobead.3


title: ChIP_501mel_set1_dCas9_ULK4_sgint31_Cas9

description: Library name: Sample 20

data_processing:
ChIP-seq reads were aligned to hg19 using BWA (v0.7.17-r1188). Unmapped and low MAPQ reads (< 30) were filtered out using SAMtools (v1.9) and PCR duplicates were removed using Picard tools (v2.18.16). Depth normalized bigWig tracks were generated using deepTools (v3.1.1) bamCoverage with `bamCoverage -b ${input_bam} -o ${output_bigwig} --normalizeUsing RPGC --ignoreForNormalization chrX chrM -bl hg19-blacklist.v2.bed`.
Assembly: hg19
Supplementary files format and content: bigWig

growth_protocol: All cell lines were cultured in media supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. All cells were cultured at 37 ºC in 5% CO2.

extract_protocol:
Cas9 ChIP was performed with 1% formaldehyde fixation for 15 min. Cells were washed once with PBS, resuspended in LB1 [50 mM HEPES (pH 7.5), 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100 and 1× complete EDTA-free protease inhibitor] and incubated with rotation at 4 °C for 10 min. Samples were pelleted, resuspended in LB2 [10 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA and 1× complete protease inhibitor] and incubated rotating at 4 °C for 10 min. Nuclei were pelleted, resuspended in LB3 with 0.2% SDS [0.2% SDS, 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100 and 1× complete protease inhibitor] and sonicated using Covaris E220 focused ultrasonicator (peak power = 150 W, duty factor = 2%, cycle per burst = 200) for 20 min. Soluble chromatin extracts were cleared by centrifugation and 1% was kept as input DNA. Sheared chromatin was diluted to contain 0.1% SDS for anti-Cas9 antibody (Active Motif, 61957) binding. 20 ul Pierce ChIP-grade magnetic beads were used for precipitation at 4 °C for 6 hr. Beads were washed 3x LiCl buffer [250 mM LiCl, 10 mM Tris-HCl (pH 8.0), 1% sodium deoxycholate, 1% NP-40 and 1 mM EDTA], 1x RIPA wash buffer [500 mM LiCl, 50 mM HEPES-KOH (pH 7.6), 0.7% sodium deoxycholate, 1% NP-40 and 1 mM EDTA] and 1x TE [10 mM Tris-HCl (pH 8.0) and 1 mM EDTA] + 50 mM NaCl. Chromatin was eluted in elution buffer [1% SDS, 50 mM Tris-HCl (pH 8.0) and 10 mM EDTA] and incubated at 65 °C for 30 min. Eluate was reverse cross-linked at 65 °C overnight, treated with RNaseA (Roche) at 37 °C for 1 hr and Proteinase K (Roche) at 55 °C for 2 hr before recovered using a miniElute PCR purification kit from Qiagen.
Library was prepared using KAPA HTP library kit (KAPA biosystems).
ChIP libraries were sequenced to obtain 30-40 M reads for each sample on NovaSeq 6000 with PE100.

treatment_protocol: dCas9-KRAB positive cells that were transduced with lentivirus carrying puromycin-resistant shRNA plasmids were treated with 2 µg/ml puromycin.

value: dCas9-KRAB, sgULK4 int31

tag: antibody

value: Cas9 antibody


title: ChIP_501mel_set2_dCas9_ULK4_sgint31_Cas9

description: Library name: Sample 21


title: RNAseq_501mel shLuc 6D rep3

description: Library name: Sample 1


title: ChIP_501mel_set2_dCas9_ULK4_sgCtrl2_Cas9

description: Library name: Sample 17

value: dCas9-KRAB, sgULK4 Ctrl 2


title: ChIP_501mel_set1_dCas9_ULK4_sgCtrl2_Cas9

description: Library name: Sample 16


title: ChIP_501mel_set2_dCas9_ULK4_sgint30_Cas9

description: Library name: Sample 19

value: dCas9-KRAB, sgULK4 int30


title: LU120_sp_p4-2

description:
Library name: LU120_sp_p4-2
Column name in RNAseq_logTPM.csv: LU120_sp_p4.2


title: ChIP_501mel_set1_dCas9_ULK4_sgint30_Cas9

description: Library name: Sample 18


title: ChIP_501mel_set2_dCas9_NOsg_Cas9

description: Library name: Sample 12


title: ChIP_501mel_set2_dCas9_ULK4_sgCtrl1_Cas9

description: Library name: Sample 15

value: dCas9-KRAB, sgULK4 Ctrl 1


title: ChIP_501mel_set1_dCas9_ULK4_sgCtrl1_Cas9

description: Library name: Sample 14


title: 501mel_dCas9_input

description: Library name: Sample 13

value: input


title: HepG2 cells, lytac1, 48 hours

description: Library name: lytac1


title: ChIP_501mel_set1_dCas9_ALK_sgint19_Cas9

description: Library name: Sample 9

value: dCas9-KRAB, sgALK int19


title: ChIP_501mel_set1_dCas9_ALK_sgint4_Cas9

description: Library name: Sample 7

value: dCas9-KRAB, sgALK int4