title: T1-3(basal diet + 0.5% succinate)


title: Participant 08, timepoint 6

status: Public on Jun 12 2023

type: SRA

city: Durham

first: Robert

last: Moseley

address: 124 Science Drive

country: USA

institute: Duke University

description: ex vivo whole blood

library_source: transcriptomic

data_processing:
Each participant's set of Fastq files were aligned to human and parasite genome reference files using STAR (version 2.7.5c)  and quantified using RSEM (version 1.3.3)
PlasmoDB-50_PvivaxP01.gff was first converted to a .gtf file using the new code scripts created for this study (see Key resources table of the manuscript)
The STAR command used for building the human STAR index was STAR --runThreadN <int> --runMode genomeGenerate --genomeDir <output_directory> --genomeFastaFiles <reference_sequence_file> --sjdbGTFfile <annotation_file>
The command for building the parasite STAR index was STAR --runThreadN <int> --runMode genomeGenerate --genomeSAindexNbases 11 --genomeDir <output_directory> --genomeFastaFiles <reference_sequence_file> --sjdbGTFfile <annotation_file>
The RSEM command, which was run once for each organism, used for building the human and parasite RSEM index was rsem-prepare-reference --num-threads <int> --gtf <annotation_file> <reference_sequence_file> <output_directory>
The STAR command for aligning reads from a single Fastq file to the human genome was STAR --runThreadN <int> --runMode alignReads --genomeDir <human_STAR_index> --readFilesIn <Fastq_file> --quantMode TranscriptomeSAM --outSAMtype BAM Unsorted --readFilesCommand zcat --outFilterType BySJout --outFileNamePrefix <output_directory> --outFilterIntronMotifs RemoveNoncanonical
The STAR command for aligning reads from a single Fastq file to the parasite genome was STAR --runThreadN <int> --runMode alignReads --genomeDir <parasite_STAR_index> --readFilesIn <Fastq_file> --quantMode TranscriptomeSAM --outSAMtype BAM Unsorted --readFilesCommand zcat --outFilterType BySJout --alignIntronMin 10 --alignIntronMax 3000 --outFileNamePrefix <output_directory> --outFilterIntronMotifs RemoveNoncanonical
Reads from a single Fastq file could map to both organisms' genomes, which can result in inaccurate quantification results. To remove the small number of reads that map to both organisms' genomes, we identified reads that were present in both the human- and parasite-associated BAM files generated from aligning a single Fastq file using STAR. These reads were then removed from both BAM files, producing new human- and parasite-associated BAM files that contained reads uniquely mapped to the respective organism. These new BAM files were used in the quantification step.
The RSEM command for quantification of the reads uniquely aligned to the human genome was rsem-calculate-expression -p <int> --bam --strandedness reverse --fragment-length-mean <avg_fragment_length> --fragment-length-sd <fragment_lenght_std_dev> --no-bam-output <filtered_BAM_file> <human_RSEM_index> <output_directory>
The RSEM command for quantification of the reads uniquely aligned to the parasite genome was rsem-calculate-expression -p <int> --strandedness reverse --fragment-length-mean <avg_fragment_length> --fragment-length-sd <fragment_lenght_std_dev> --bam --no-bam-output <filtered_BAM_file> <parasite_RSEM_index> <output_directory>
Alignment and quantification was performed on the Duke Compute Cluster (https://rc.duke.edu/dcc/) at Duke University which, at the time of use, used CentOS 8 as the operating system and SLURM as the scheduler for the entire system.
Genes which appear in the comprehensive gene annotation file but which were not expressed (i.e. 0 TPM units) at any time point by a participant or parasite population were removed.
Within the GENCODE comprehensive gene annotation file used for this study are 44 genes with duplicate copies in the pseudoautosomal regions on the X and Y chromosomes (Table S2 of manuscript). Since each of these 44 pairs contained two copies of the same gene, quantification resulted in 44 pairs of duplicated expression profiles for each study participant. Because each participant is male, we added together the duplicate expression profiles in the 44 pairs and retained only a single copy of the duplicated genes. In this way we ensured that pseudoautosomal genes could not have an outsized influence in downstream analyses (e.g. circadian phase estimates) if these genes were found to be highly periodic and sufficiently highly expressed.
QuIn this study, species-specific Quantile Normalization was independently applied to each participant's samples and each participant's parasite samples. In particular, genes within each sample (participant, organism, and time point) were sorted according to their TPM values, thereby ranking genes from least expressed to most expressed in each organism and time point. The average TPM value of all genes occupying the same rank in these sorted lists was then computed across all timepoints within each participant and organism and the original TPM value of each gene was replaced with the average of all genes occupying it's same rank. Here care was taken to include all equally-ranked genes, including those which are ambiguously ranked because they have identical TPM values within a sample. Finally, each sample was reordered by gene, to realign the now between-sample normalized expression levels.
Sample 18's hour-24 time point sample was accidentally destroyed prior to RNA sequencing. Expression levels for this time point were interpolated following Quantile Normalization using Piecewise Cubic Hermite Interpolating Polynomial (PCHIP) interpolation.
Assembly: human: gencode.GRCh38.primary_assembly.genome.fa ; parasite: PlasmoDB-50_PvivaxP01_Genome.fasta
genome feature file: human: gencode.v36.primary_assembly.annotation.gtf; parasite: PlasmoDB-50_PvivaxP01.gff
Supplementary files format and content: Each Sample, e.g., Sample 02, has a tab-delimited text file containing quantile normalized TPM values for each sampling time point.
Supplementary files format and content: counts_human.tsv and counts_vivax.tsv files contain the expected read counts as determined by RSEM for each gene.

molecule: total RNA

organism:
Plasmodium vivax
Homo sapiens

source_name: whole blood

growth_protocol: 18 ml of blood was drawn from each study participant into heparin tubes for the ex vivo time series experiment. 3.2 ml of the sample was mixed with 156.8 ml of McCoy’s 5A culture media supplemented with 2 mM of L-glutamine, 20% AB serum, generating a 2% haematocrit sample. To collect the culture sample at 3 hour intervals over the subsequent 48 hours, 10 ml of the culture mixture were aliquotted into 25 cm2 culture flasks (16 flasks in total for each study participant). Cultures were gassed with 5% CO2, 5% O2, 90% N2 gas and placed in an incubator at 37°C. Approximately every 3 hours, one of the culture flasks was removed, and media and cell pellets were kept in separate tubes and placed immediately in liquid nitrogen, for a total of 17 time point samples including the time 0 sample.

extract_protocol:
Frozen cell pellets were used for RNA extraction using the Agencourt RNAdvance Blood RNA purification kit and associated protocols. Samples were cleaned before sequencing with Zymo Research OneStepTM PCR Inhibitor Removal Kit.
Libraries were constructed using a kit specific for blood that depleted both ribosomal and globin RNAs.

tag: tissue

value: whole blood

tag: disease state

value: Plasmodium vivax infected

tag: time

value: hr 15

tag: study participant


title: jcov61-cd34_S31_L002

city: New York

first: Jin-Gyu

last: Cheong

address: 1300 York Avenue

institute: Weill Cornell Medicine

description:
PCR amplicon
CD34_CP.csv

library_source: genomic

data_processing:
Paired-end Bulk ATAC-seq reads were quality trimmed using Trimmomatic.
trimmed reads were aligned to human genome (hg38) using BWA
After preprocessing and quality filtering, peaks were called on alignments with MACS2 using the BAMPE option.
For differential accessibility analyses the consensus peak set was generated using the R package DiffBind. The consensus peakset was generated per celltype.
Peaks only present in at least two samples were included in the analysis. Raw read counts were normalized within the cinaR package.
Assembly: hg38
Supplementary files format and content: Normalized counts for Atacseq consensus peaks per tissue/cell type

molecule: genomic DNA

organism: Homo sapiens

source_name: PBMC

extract_protocol: As previously described in Corces, M.R. et al. 2017, PMID: 28846090

value: PBMC

tag: cell type

value: HSPC

tag: age (in_years)

tag: Sex

value: Female

tag: clinical status


title: C-2(basal diet)

city: Yinchuan

first: Wenyi

last: Ren

address: 489 Helanshan West Road, Xixia District, Yinchuan City, Ningxia

department: College of Animal Science and Technology

country: China

institute: Ningxia University

data_processing:
Illumina Casava1.7 software used for basecalling.
we use Cutadapt(v1.15) software to filter the sequencing data to get high quality sequence（Clean Data）forfurther analysis .
Thefiltered reads were mapping to the reference genome using HISAT2 v2.1.0
we used HTSeq(0.9.1) statistics to compare the Read Count values on each gene as the originalexpression of the gene, and then used FPKM to standardize the expression.
Assembly: Genome:GCF_016772045.1_ARS-UI_Ramb_v2.0_genomic.fna    Path:https://www.ncbi.nlm.nih.gov/assembly/GCF_016772045.1  Genebuild by:NCBI
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample

organism: Ovis aries

source_name: Tan sheep

extract_protocol:
Total RNA was isolated using the Trizol Reagent (Invitrogen Life Technologies)
Select total RNA >=1ug and use the NEBNext Ultra II RNA Library Prep Kit for Illumina

tag: age

value: 5 month-old

tag: treatment

value: basal diet


title: Blood_Platelets_MGH-NSCLC-L117-TR686

city: Amsterdam

first: Mafalda

last: Antunnes

state: zuid holland

address: de boelelaan 1118

department: NRG

country: Netherlands

institute: Amsterdam UMC

data_processing:
Single-end 100 bp reads were subjected to pre-aligment 5'-end quality trimming and sequence adapter clipping using Trimmomatic.
Cleaned-up reads were mapped to the hg19 reference genome using STAR (version 2.3.0), using splice aware alignments and allowing for 10 mismatches.
Intron-spanning read summarization was performed using a samtools-based filtering step and HTSeq (version 0.6.1) using union-mode, unstranded reads and minimal mapping quality of 35, all guided by the Ensembl gene annotation version 75.
Assembly: hg19
Supplementary files format and content: Samples in the Rdata-file were processed according to the described protocol (after filtering for low-abundent RNAs).

source_name: Thrombocytes

extract_protocol:
Blood platelets were isolated from whole blood in purple-cap BD Vacutainers containing the EDTA anti-coagulant. The cells and aggregates were removed by centrifugation at room temperature for 20 minutes at 120g, resulting in platelet-rich plasma. The platelets were isolated from the platelet-rich plasma by centrifugation at room temperature for 20 minutes at 360g. The platelet pellet was collected in 30 μl RNAlater (Life Technologies), incubated overnight at 4°C and frozen at -80°C for further use. Frozen platelets were thawed on ice and total RNA was isolated using the mirVana RNA isolation kit (Life Technologies) according to the manufacturers’ protocol. Complementary purification of small RNAs was included in the isolation procedure by addition of miRNA homogenate (Life Technologies). Total RNA was dissolved in 30 μl Elution Buffer (Life Technologies) and RNA quality and quantity was measured using Bioanalyzer 2100 with RNA 6000 Picochip (Agilent).
100-500 pg of platelet total RNA diluted in nuclease free H2O was subjected to cDNA synthesis and amplification using the SMARTer Ultra Low RNA Kit for Illumina Sequencing v3 (Clontech, cat. nr. 634853) according to the manufacturers’ protocol. Conversion and efficient amplification of cDNA was quality-controlled using the Bioanalyzer 2100 with DNA High Sensitivity chip (Agilent). Samples with detectable fragments in the 300-7500 bp region were selected for further processing and Covaris shearing by sonication (Covaris Inc). Sample preparation for Illumina sequencing was performed using the Truseq DNA Sample Preparation Kit (Illumina, cat nr. FC-121-2001) or Truseq Nano DNA Sample Preparation Kit (Illumina, cat nr. FC-121-4001). Sample quality and quantity was measured using the DNA 7500 chip or DNA High Sensitivity chip (Agilent). High-quality samples with product sizes between 300-500 bp were pooled in equimolar concentrations and submitted for 100 bp Single Read sequencing on the Hiseq 2500 or 4000 platform (Illumina).

value: Thrombocytes

tag: cancer stage

tag: patient group

value: NSCLC


title: Blood_Platelets_Vumc-NSCLC-179-TR670


title: jcov61-cd14_S23_L002

description:
PCR amplicon
CD14_CP.csv

value: CD14 Monocytes


title: C-1(basal diet)


title: Jcov47-cd34_S40_L001

description:
PCR amplicon
CD34_CP.csv

value: Male


title: Participant 08, timepoint 5

description: ex vivo whole blood

value: hr 12


title: Blood_Platelets_Vumc-NSCLC-173-TR668


title: Participant 08, timepoint 4

description: ex vivo whole blood

value: hr 9


title: Blood_Platelets_Vumc-NSCLC-172-TR667


title: jcov47-cd14_S18_L001

description:
PCR amplicon
CD14_CP.csv


title: jcov88-cd14_S30_L002

description:
PCR amplicon
CD14_CP.csv


title: Wild type Cortex

city: Shanghai

first: Guoping

last: Liu

address: 138 Yi Xue Yuan Road

institute: Fudan University

description:
Wild type littermate
SHH.csv

library_source: transcriptomic single cell

data_processing:
cellranger mkfastq demultiplexes raw base call (BCL) files generated by Illumina sequencers into FASTQ files.
cellranger count takes FASTQ files from cellranger mkfastq and performs alignment, filtering, barcode counting, and UMI counting.
Stringtie (vetrsion: 1.3.0) was used to count the read numbers mapped to each gene, and standardrized the counts using TMM (Trimmed mean of M values)
cellranger aggr aggregates outputs from multiple runs of cellranger count, normalizing those runs to the same sequencing depth and then recomputing the feature-barcode matrices and analysis on the combined data.
Finally, Seurat (V3) were used for the following dimensionality reduction, clustering, and gene expression.
Assembly: mm10 for mice and ASM1176430v1.1 for Ferret
Supplementary files format and content: gene count

organism: Mus musculus

source_name: E18 wild type mouse cortex

extract_protocol:
The Chromium droplet-based sequencing platform (10X Genomics) was used to generate scRNA-seq libraries, following the manufacturer’s instructions (manual document part number: CG00052 Rev C).
The Chromium droplet-based sequencing platform (10X Genomics) was used to generate scRNA-seq libraries, following the manufacturer’s instructions.

treatment_protocol: For mouse cortex: E18 wild type and Smo CKO (Flash-Tag were injected at E17, and the cortex was harvested at E18) cortices were carefully dissected under a fluorescent stereoscope, and incubated in 4 ml of papain solution for 20 min at 37℃, The cortical tissues were gently triturated, filtered through a 40 mm strainer, and washed with HBSS to obtain the single cell suspension. The wild type E39 ferret cortex were dissected and the treatment methods are the same as those of mice.

tag: strain

value: Cortex

tag: developmental stage

value: Embryonic day 18


title: Participant 08, timepoint 2

description: ex vivo whole blood

value: hr 3


title: Blood_Platelets_Vumc-NSCLC-170-TR666


title: Blood_Platelets_Vumc-NSCLC-183-TR671


title: Smo CKO Cortex

description:
Smo CKO littermate
SHH.csv

source_name: E18 Smo CKO mouse cortex


title: Blood_Platelets_Vumc-LGG-VU106-1-TR605


title: jcov107-cd14_S16_L001

description:
PCR amplicon
CD14_CP.csv

value: nonCov


title: Participant 08, timepoint 3

description: ex vivo whole blood

value: whole blood

value: Plasmodium vivax infected

value: hr 6


title: jcov103-cd34_S36_L001

description:
PCR amplicon
CD34_CP.csv

value: PBMC

value: HSPC


title: jcov108-cd14_S39_L001

description:
PCR amplicon
CD14_CP.csv


title: Participant 08, timepoint 1

description: ex vivo whole blood

value: hr 0


title: Aorta, WT, scRNAseq

first: zixin

last: liu

address: 320 Yueyang road, Life Science Research Building A2112

institute: Center for Excellence in Molecular Cell Science

description: 10x Genomics

data_processing:
Each scRNA-seq data set was processed by CellRanger (v4.0.0) pipeline to perform the alignment against the modified mouse reference genome mm10, filter the alignment and count barcodes and UMIs
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files

source_name: Aorta

extract_protocol:
ProTracer mice were treated with tamoxifen at 8 weeks old. Then 8 weeks later, aortas were harvested from the mice, perfused with cold PBS, and then finely minced and transferred to the digestion mix (HBSS, 1U/µl Collagenase type 1 (LS004197, Worthington)). Tissues were incubated with the digestion mix at 37℃ for 30 minutes. The cell suspension was filtered through a 70 µm cell strainer and then pelleted by centrifugation at 300g for 5 minutes. After washing cells with 500ml HBSS, cells were loaded onto the 10x Chromium controller the generate Gel Bead-in-Emulsions (GEMs), which were further processed into single cell 5’ gene expression libraries using Chromium Single Cell 5’ Library Kit (v1.1).
The cell suspense was loaded in 10x Chromium controller to generate GEMs, which were further processed into single cell 5ʹ gene expression libraries using Chromium Single Cell 5ʹ Library Kit (v1.1).

value: Aorta

value: Aortic cell

tag: genotype

value: R26-DreER, Ki67-CrexER, R26-GFP

value: tamoxifen at 8 weeks old, and harvested after 8 weeks


title: jcov102-cd34_S35_L001

description:
PCR amplicon
CD34_CP.csv

value: Male


title: Participant 02, timepoint 15

description: ex vivo whole blood

value: hr 42


title: Blood_Platelets_MGH-NSCLC-L40-TR520

value: Thrombocytes

value: NSCLC


title: jcov102-cd14_S12_L001

description:
PCR amplicon
CD14_CP.csv

value: CD14 Monocytes


title: PA14_DrsaLDlasR_2_rsls_A

city: Braunschweig

first: Matthias

last: Preusse

address: Inhoffenstr. 7

country: Germany

institute: Helmholtz Centre for Infection Research

description:
start OD600 of 0.05; treated for 2 h with 0.1 mM IPTG  at OD (0.5-0.7)
rpg_table_PA14_pSEVA_constructs.csv

data_processing:
Pseudomonas aeruginosa UCBPP-PA14
Reads were mapped using bowtie2 (--very-sensitive-local --no-mixed --no-discordant --fr )
Reference genome: NC_008463.1
Files were converted into bam format using samtools
reads per gene from Pseudomonas aeruginosa UCBPP-PA14 were extracted using featureCounts (-Q 20 --fracOverlap 0.5)
Supplementary_files_format_and_content: reads per gene include antisense mapped reads of lasR and rsaL (using featureCounts options -s 1/2), antisense reads were selected using R environent

organism: Pseudomonas aeruginosa UCBPP-PA14

source_name: RNASeq of planctonic culture of Pseudomonas aeruginosa UCBPP-PA14 delta rsaL delta lasR pSEVA224::rsaL_short pSEVA644::lasR_short treated for 2 h with 0.1 mM IPTG  at OD (0.5-0.7)

growth_protocol: Bacterial subcultures were grown in liquid LB, at 37 °C and 180 rpm shaking. For cells harboring plasmid pJN105, medium was supplemented with gentamicin (50 µg/ml). For cells harboring plasmids pSEVA224 and pSEVA644, medium was supplemented with gentamicin (40 µg/ml) and kanamycin (200 µg/ml).

extract_protocol:
Bacterial cell pellets were resuspended in RNAprotect. RNA extraction was done as previously described (Schinner et al., 2020)
NEBNext Ultra II Directional RNA Library Prep

treatment_protocol: Cells harboring plasmid pJN105 were treated with 0.1 % arabinose at OD600 of 1 and harvested at OD600 of 2. Cells harboring plasmids pSEVA224 and pSEVA644 were treated with 0.1 mM IPTG at OD600 of 0.5-0.7 and harvested 2 h later at OD600 of 1.1-1.6.

tag: cell line/strains


title: PA14_DrsaLDlasR_1_emem_B

source_name: RNASeq of planctonic culture of Pseudomonas aeruginosa UCBPP-PA14 delta rsaL delta lasR  pSEVA224 pSEVA644 treated for 2 h with 0.1 mM IPTG  at OD (0.5-0.7)


title: Participant 02, timepoint 14

description: ex vivo whole blood

value: hr 39


title: Blood_Platelets_Vumc-NSCLC-151-TR463


title: PA14_DrsaLDlasR_2_rsls_B

value: PA14 delta rsaL delta lasR pSEVA224::rsaL_short pSEVA644::lasR_short


title: HDw-cd14_S10_L001

description:
PCR amplicon
CD14_CP.csv

value: Healthy


title: Blood_Platelets_Vumc-NSCLC-129-TR457


title: Blood_Platelets_MGH-NSCLC-L07-TR459


title: PA14_DrsaLDlasR_4_rsll_A

source_name: RNASeq of planctonic culture of Pseudomonas aeruginosa UCBPP-PA14 delta rsaL delta lasR  pSEVA224::rsaL_short pSEVA644::lasR_long treated for 2 h with 0.1 mM IPTG  at OD (0.5-0.7)


title: Participant 02, timepoint 12

description: ex vivo whole blood

value: whole blood

value: Plasmodium vivax infected

value: hr 33


title: HDq-cd14_S29_L001

description:
PCR amplicon
CD14_CP.csv

value: PBMC

value: Female


title: Participant 02, timepoint 13

description: ex vivo whole blood

value: hr 36


title: HDt-cd14_S32_L001

description:
PCR amplicon
CD14_CP.csv


title: PA14_DrsaLDlasR_1_emem_A

value: PA14 delta rsaL delta lasR  pSEVA224 pSEVA644


title: Blood_Platelets_Vumc-NSCLC-VU146-TR374


title: Blood_Platelets_MGH-NSCLC-L58-TR525


title: PA14_DrsaLDlasR_4_rsll_B

value: PA14 delta rsaL delta lasR  pSEVA224::rsaL_short pSEVA644::lasR_long


title: PA14_DrsaLDlasR_5_rslas_B

source_name: RNASeq of planctonic culture of Pseudomonas aeruginosa UCBPP-PA14 delta rsaL delta lasR  pSEVA224::rsaL_short pSEVA644::lasR_antisense treated for 2 h with 0.1 mM IPTG  at OD (0.5-0.7)


title: Participant 02, timepoint 11

description: ex vivo whole blood

value: hr 30


title: Blood_Platelets_MGH-NSCLC-L-89


title: Blood_Platelets_MGH-NSCLC-L-09


title: Participant 02, timepoint 10

description: ex vivo whole blood

value: hr 27


title: HDp-cd34_S28_L001

description:
PCR amplicon
CD34_CP.csv

value: HSPC


title: PA14_DrsaLDlasR_7_rlls_B

source_name: RNASeq of planctonic culture of Pseudomonas aeruginosa UCBPP-PA14 delta rsaL delta lasR  pSEVA224::rsaL_long pSEVA644::lasR_short treated for 2 h with 0.1 mM IPTG  at OD (0.5-0.7)


title: OCT4-GFP positive germ cells, GR WT control, biological rep 3

city: San Francisco

first: Steven

middle: Anthony

last: Cincotta

state: California

address: 35 Medical Center Way

institute: UCSF

description:
FFM0006
Tag-Seq_featureCounts_aggregate_female.csv
Female-DEL_vs_Female-WT.csv

data_processing:
Raw fastq reads were processed to remove adapter sequences, reads containing poly-N, and low quality reads
Reads were aligned to mm10 reference genome using STAR v2.6.0
Counts of reads mapping to each gene were determined using featureCounts v1.6.3
Pairwise differential expression analysis was performed using edgeR v3.28.1 and limma v3.42.2. P-values were adjusted using Benjamini and Hochber FDR.
Assembly: mm10
Supplementary files format and content: CSV file containing raw counts for each sample
Supplementary files format and content: CSV files containing pairwise differential gene expression output from edgeR/limma

molecule: polyA RNA

source_name: OCT4-GFP positive germ cells sorted from E17.5 ovary

extract_protocol:
RNA was harvested using RNeasy Micro Kit (QIAgen)
Library preparations and sequencing were performed by the University of California, Davis DNA Technologies & Expression Analysis Core. Gene expression profiling was carried out using a 3’ Tag-RNA-Seq protocol. Barcoded sequencing libraries were prepared using the QuantSeq 3’ mRNA-Seq Library Prep FWD kit (Lexogen) for multiplexed sequencing according to the manufacturer recommendations. The library fragment size distribution was determined using microcapillary gel electrophoresis on a Bioanalyzer 2100 (Agilent), and libraries were quantified using a Qubit fluorometer (LifeTechnologies). Final libraries were pooled in equimolar ratios and sequenced on an Illumina HiSeq 4000.

treatment_protocol: For both full body knockout and conditional deletion of GR, embryos were dissected from pregnant dams the morning of E17.5 into cold 0.4% BSA in PBS, and tail clips were taken from each embryo to determine GR genotype. After removing mesonephroi, fetal ovaries from individual embryos were digested for FACS sorting using 0.25% Trypsin-EDTA at 37°C for 30 min, with gentle pipetting every 10-15 minutes to facilitate dissociation. After 30 minutes, DNase I (1 mg / mL) was added at a 1:10 dilution, and samples were incubated another 10 minutes at 37°C. Samples were pipetted to ensure complete digestion, and then an equal volume of ice-cold FBS was added to inactivate trypsin. To prepare for FACS sorting, Sytox Blue viability dye was added to samples at 1:1000 dilution, and then samples were filtered through a 35 μm filter into FACS tubes. Samples were FACS sorted on a BD FACSAria II, where Tg:Oct4-GFP+ germ cells from individual embryo ovary pairs were sorted directly into QIAgen RLT+ buffer and stored at -80 until ready for RNA extractions.

value: ovary

value: OCT4-GFP positive germ cells

value: female

tag: mouse strain

value: Tg(Pou5f1-EGFP)2Mnn Nr3c1tm1.1Jda


title: OCT4-GFP positive germ cells, GR WT control, biological rep 2

description:
FFM0002
Tag-Seq_featureCounts_aggregate_female.csv
Female-DEL_vs_Female-WT.csv

tag: tissue


title: PA14_DrsaLDlasR_8_rasls_A

source_name: RNASeq of planctonic culture of Pseudomonas aeruginosa UCBPP-PA14 delta rsaL delta lasR  pSEVA224::rsaL_antisense pSEVA644::lasR_short treated for 2 h with 0.1 mM IPTG  at OD (0.5-0.7)


title: Hdi-cd14_S19_L002

description:
PCR amplicon
CD14_CP.csv

value: CD14 Monocytes


title: PA14_DrsaLDlasR_7_rlls_A

value: PA14 delta rsaL delta lasR  pSEVA224::rsaL_long pSEVA644::lasR_short


title: Participant 02, timepoint 9

description: ex vivo whole blood

value: whole blood

tag: disease state

value: hr 24


title: OCT4-GFP positive germ cells, GR WT control, biological rep 1

description:
FFM0001
Tag-Seq_featureCounts_aggregate_female.csv
Female-DEL_vs_Female-WT.csv


title: cov201-cd14_S18_L002

description:
PCR amplicon
CD14_CP.csv

value: Male


title: Participant 02, timepoint 6

description: ex vivo whole blood

value: Plasmodium vivax infected

tag: time

value: hr 15


title: EFNB2-D62Q library sorted for low NiV-G binding (Replicate 2; PCR Fragment A Fragment B)

city: Urbana

first: Erik

last: Procko

state: IL

address: 600 S Matthews Ave RAL 311

department: Biochemistry

institute: University of Illinois, Urbana-Champaign

data_processing:
Data was analyzed using python scripts in the Enrich package: http://depts.washington.edu/sfields/software/enrich/
Fuser command (Fragment A): python3 Paired_read_fuser.py --path directory_A/ --paired_mismatch_threshold 250 --read1 sample_R1.fastq --read2 sample_R2.fastq --wtseq ATCGTGTTGGAGCCCATATATTGGAATAGCTCCAATTCAAAATTTCTGCCAGGACAGGGACTCGTTCTCTATCCTCAAATCGGCGACAAATTGCAGATTATATGCCCTAAGGTTGACTCTAAGACTGTTGGGCAGTATGAGTATTATAAAGTGTATATGGTAGATAAGGATCAGGCGGATCGCTGTACTATCAAAAAA --read1_overlap_start 49 --read1_overlap_end 247 --read2_overlap_start 31 --read2_overlap_end 229 --mode B
Fuser command (Fragment B): python3 Paired_read_fuser.py --path directory_B/ --paired_mismatch_threshold 250 --read1 sample_R1.fastq --read2 sample_R2.fastq --wtseq GAGAATACGCCTCTTCTGAACTGTGCGAAACCCGATCAAGATATTAAATTTACAATTAAGTTTCAAGAATTTTCACCTAATTTGTGGGGTCTTGAGTTCCAGAAGAACAAAGACTATTACATCATTAGCACCTCCAACGGAAGCCTTGAGGGTCTGGATAATCAGGAAGGTGGAGTTTGTCAAACTCGCGCCATGAAAATTCTGATGAAGGTAGGA --read1_overlap_start 24 --read1_overlap_end 240 --read2_overlap_start 1 --read2_overlap_end 217 --mode B
Aligner command (Fragment A): python3 Fused_read_aligner.py --path directory_A/ --infile sample_R1.fast_B_qc1 --referenceDNA ATCGTGTTGGAGCCCATATATTGGAATAGCTCCAATTCAAAATTTCTGCCAGGACAGGGACTCGTTCTCTATCCTCAAATCGGCGACAAATTGCAGATTATATGCCCTAAGGTTGACTCTAAGACTGTTGGGCAGTATGAGTATTATAAAGTGTATATGGTAGATAAGGATCAGGCGGATCGCTGTACTATCAAAAAA --referenceAA IVLEPIYWNSSNSKFLPGQGLVLYPQIGDKLQIICPKVDSKTVGQYEYYKVYMVDKDQADRCTIKK --gap_max 250 --unresolvable_max 250 --maxmutrun 250 --avg_quality 30 --chaste 1 --Ncount_max 250 --use_N 0 --mode B
Aligner command (Fragment B): python3 Fused_read_aligner.py --path directory_B/ --infile sample_R1.fast_B_qc1 --referenceDNA GAGAATACGCCTCTTCTGAACTGTGCGAAACCCGATCAAGATATTAAATTTACAATTAAGTTTCAAGAATTTTCACCTAATTTGTGGGGTCTTGAGTTCCAGAAGAACAAAGACTATTACATCATTAGCACCTCCAACGGAAGCCTTGAGGGTCTGGATAATCAGGAAGGTGGAGTTTGTCAAACTCGCGCCATGAAAATTCTGATGAAGGTAGGA --referenceAA ENTPLLNCAKPDQDIKFTIKFQEFSPNLWGLEFQKNKDYYIISTSNGSLEGLDNQEGGVCQTRAMKILMKVG --gap_max 250 --unresolvable_max 250 --maxmutrun 250 --avg_quality 30 --chaste 1 --Ncount_max 250 --use_N 0 --mode B
MapCounts command: python3 mapCounts.py --path directory/ --infile sample_R1.fast_B_qc1_PRO_qc2
MapRatios command: python3 mapRatios.py --path directory/ --templatepath /deepseq_scripts/r_deepseq_scripts/ --infile2 mapcounts_library --infile1 mapcounts_sorted
MapParts command: python3 mapParts.py --path directory/ --infile mapratios_sorted_library --mode mutations:1
Unlink script: python3 mapUnlink.py --path directory/ --infile mapratios_sorted_library.m1 --type protein --mode ratios --size number_of_residues_in_fragment
In Excel, the log(base2) enrichment ratio of the wildtype sequence was subtracted from the log(2) enrichment ratios for all single mutations.
Assembly: Not applicable
Supplementary files format and content: Excel spreadsheet of log(base2) enrichment ratios for each single amino acid substitution.  Also includes the total reads for each mutation in the naive plasmid library.

source_name: Synthetic coding sequences

extract_protocol:
Total RNA was extracted from FACS sorted cells using a GeneJet kit (Thermo Scientific), and cDNA was synthesized using high fidelity Accuscript (Agilent) primed from one of two gene-specific primers.  The EFNB2-D62Q cDNA was PCR amplified as two separate fragments that together fully cover the diversified regions of EFNB2-D62Q present in the SSM library.  The PCR fragments were amplified for Illumina sequencing in two rounds.  In the first round, primer overhangs added sequences for annealing to the Illumina sequencing primers. In the second round, primer overhangs added unique barcodes and Illumina adaptors.  To determine the frequencies of mutations in the naive library, the library plasmid DNA was also used as a template for PCR amplification and Illumina sequencing.
Primer used for reverse transcription with Accuscript for all fragments: pCEP4_EBV_Reverse (GTGGTTTGTCCAAACTCATC)
Primer pair for 1st round of PCR (Fragment A): MiSeq_CCR5_myctag_F (TCTTTCCCTACACGACGCTCTTCCGATCTCGAGCAAAAGCTGATCTCAG) and Illumina_EFNB2_295_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTTCAGAAGAGGCGTATTCTC)
Primer pair for 1st round of PCR (Fragment B): Illumina_EFNB2_271_F (TCTTTCCCTACACGACGCTCTTCCGATCTGCGGATCGCTGTACTATCAA) and Illumina_EFNB2_525_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATTCCGCGTTGATCCCGCT)
Primer pair for 2nd round of PCR (added I5 and I7 Illumina adaptors): MiSeq_Start_Adaptamer (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT) and MiSeq_Index_Adaptamer (CAAGCAGAAGACGGCATACGAGAT-6nt-barcode-GTGACTGGAGTTCAGACGTGTGCTCTTC)
Amplicons were deep sequenced on a NovaSeq 6000 using a 2x250 nt paired end protocol.

treatment_protocol: Expi293F cells were transfected using expifectamine (ThermoFisher) with a pCEP4 episomal plasmid-based library encoding variants of myc-tagged ENFB2-D62Q diluted with a 1500-fold excess of carrier DNA; these transfection conditions ensure that most cells express no more than a single EFNB2-D62Q sequence variant, thereby linking genotype to phenotype.  Cells were harvested 24 h post-transfection and co-incubated with anti-myc Alexa 647 (1/250 dilution) and 1 uM sEphB3-Fc and 1 uM sEphB4-Fc.  Cells were washed, incubated with cleared medium from cells secreting NiV-G-sfGFP (1/5 dilution), washed again, and sorted by FACS.  Gates were drawn for the main population of viable cells using forward and side scatter properties and exclusion of the fluorescent viability dye propidium iodide.  The EFNB2-positive cells based on Alexa 647 fluorescence were further gated to collect the top 10% and bottom 15% of cells for bound NiV-G-sfGFP relative to EFNB2 expression.

tag: protein

tag: sort gate

value: Bottom 15% of cells for bound NiV-G-sfGFP relative to surface EFNB2-D62Q expression


title: OCT4-GFP positive germ cells, GR full deletion, biological rep 2

description:
FFD0002
Tag-Seq_featureCounts_aggregate_female.csv
Female-DEL_vs_Female-WT.csv

tag: cell type


title: EFNB2-D62Q library sorted for high NiV-G binding (Replicate 1; PCR Fragment A Fragment B)

value: Top 10% of cells for bound NiV-G-sfGFP relative to surface EFNB2-D62Q expression


title: OCT4-GFP positive germ cells, GR full deletion, biological rep 3

description:
FFD0004
Tag-Seq_featureCounts_aggregate_female.csv
Female-DEL_vs_Female-WT.csv

tag: Sex


title: EFNB2-D62Q library sorted for high NiV-G binding (Replicate 2; PCR Fragment A Fragment B)


title: Participant 02, timepoint 7

description: ex vivo whole blood

value: hr 18

tag: study participant


title: PA14_DrsaLDlasR_14_rllas_A

source_name: RNASeq of planctonic culture of Pseudomonas aeruginosa UCBPP-PA14 delta rsaL delta lasR pSEVA224::rsaL_long pSEVA644::lasR_antisense treated for 2 h with 0.1 mM IPTG  at OD (0.5-0.7)


title: Participant 02, timepoint 5

description: ex vivo whole blood

value: hr 12


title: EFNB2-D62Q library sorted for low NiV-G binding (Replicate 1; PCR Fragment A Fragment B)


title: PA14_DrsaLDlasR_9_rlll_A

source_name: RNASeq of planctonic culture of Pseudomonas aeruginosa UCBPP-PA14 delta rsaL delta lasR  pSEVA224::rsaL_long pSEVA644::lasR_long treated for 2 h with 0.1 mM IPTG  at OD (0.5-0.7)


title: PA14_DrsaLDlasR_14_rllas_B

value: PA14 delta rsaL delta lasR pSEVA224::rsaL_long pSEVA644::lasR_antisense


title: PA14_DrsaLDlasR_5_rslas_A

value: PA14 delta rsaL delta lasR  pSEVA224::rsaL_short pSEVA644::lasR_antisense


title: Library of EFNB2-D62Q variants (PCR Fragment A Fragment B)

library_source: other

molecule: other

tag: molecule subtype

value: plasmid DNA


title: Participant 02, timepoint 8

description: ex vivo whole blood

source_name: whole blood

value: hr 21


title: Participant 02, timepoint 2

description: ex vivo whole blood

value: hr 3


title: OCT4-GFP positive germ cells, GR flox control, biological rep 3

description:
CFF0004
Tag-Seq_featureCounts_aggregate_female.csv
Female-cKO_vs_Female-flox.csv

value: ovary

value: OCT4-GFP positive germ cells

value: female


title: OCT4-GFP positive germ cells, GR full deletion, biological rep 1

description:
FFD0001
Tag-Seq_featureCounts_aggregate_female.csv
Female-DEL_vs_Female-WT.csv

value: Tg(Pou5f1-EGFP)2Mnn Nr3c1tm1.1Jda


title: EFNB2 library sorted for low NiV-G binding (Replicate 2; PCR Fragment A Fragment B)

data_processing:
Data was analyzed using python scripts in the Enrich package: http://depts.washington.edu/sfields/software/enrich/
Fuser command (Fragment A): python3 Paired_read_fuser.py --path directory_A/ --paired_mismatch_threshold 250 --read1 sample_R1.fastq --read2 sample_R2.fastq --wtseq ATCGTGTTGGAGCCCATATATTGGAATAGCTCCAATTCAAAATTTCTGCCAGGACAGGGACTCGTTCTCTATCCTCAAATCGGCGACAAATTGGATATTATATGCCCTAAGGTTGACTCTAAGACTGTTGGGCAGTATGAGTATTATAAAGTGTATATGGTAGATAAGGATCAGGCGGATCGCTGTACTATCAAAAAA --read1_overlap_start 49 --read1_overlap_end 247 --read2_overlap_start 31 --read2_overlap_end 229 --mode B
Fuser command (Fragment B): python3 Paired_read_fuser.py --path directory_B/ --paired_mismatch_threshold 250 --read1 sample_R1.fastq --read2 sample_R2.fastq --wtseq GAGAATACGCCTCTTCTGAACTGTGCGAAACCCGATCAAGATATTAAATTTACAATTAAGTTTCAAGAATTTTCACCTAATTTGTGGGGTCTTGAGTTCCAGAAGAACAAAGACTATTACATCATTAGCACCTCCAACGGAAGCCTTGAGGGTCTGGATAATCAGGAAGGTGGAGTTTGTCAAACTCGCGCCATGAAAATTCTGATGAAGGTAGGA --read1_overlap_start 24 --read1_overlap_end 240 --read2_overlap_start 1 --read2_overlap_end 217 --mode B
Aligner command (Fragment A): python3 Fused_read_aligner.py --path directory_A/ --infile sample_R1.fast_B_qc1 --referenceDNA ATCGTGTTGGAGCCCATATATTGGAATAGCTCCAATTCAAAATTTCTGCCAGGACAGGGACTCGTTCTCTATCCTCAAATCGGCGACAAATTGGATATTATATGCCCTAAGGTTGACTCTAAGACTGTTGGGCAGTATGAGTATTATAAAGTGTATATGGTAGATAAGGATCAGGCGGATCGCTGTACTATCAAAAAA --referenceAA IVLEPIYWNSSNSKFLPGQGLVLYPQIGDKLDIICPKVDSKTVGQYEYYKVYMVDKDQADRCTIKK --gap_max 250 --unresolvable_max 250 --maxmutrun 250 --avg_quality 30 --chaste 1 --Ncount_max 250 --use_N 0 --mode B
Aligner command (Fragment B): python3 Fused_read_aligner.py --path directory_B/ --infile sample_R1.fast_B_qc1 --referenceDNA GAGAATACGCCTCTTCTGAACTGTGCGAAACCCGATCAAGATATTAAATTTACAATTAAGTTTCAAGAATTTTCACCTAATTTGTGGGGTCTTGAGTTCCAGAAGAACAAAGACTATTACATCATTAGCACCTCCAACGGAAGCCTTGAGGGTCTGGATAATCAGGAAGGTGGAGTTTGTCAAACTCGCGCCATGAAAATTCTGATGAAGGTAGGA --referenceAA ENTPLLNCAKPDQDIKFTIKFQEFSPNLWGLEFQKNKDYYIISTSNGSLEGLDNQEGGVCQTRAMKILMKVG --gap_max 250 --unresolvable_max 250 --maxmutrun 250 --avg_quality 30 --chaste 1 --Ncount_max 250 --use_N 0 --mode B
MapCounts command: python3 mapCounts.py --path directory/ --infile sample_R1.fast_B_qc1_PRO_qc2
MapRatios command: python3 mapRatios.py --path directory/ --templatepath /deepseq_scripts/r_deepseq_scripts/ --infile2 mapcounts_library --infile1 mapcounts_sorted
MapParts command: python3 mapParts.py --path directory/ --infile mapratios_sorted_library --mode mutations:1
Unlink script: python3 mapUnlink.py --path directory/ --infile mapratios_sorted_library.m1 --type protein --mode ratios --size number_of_residues_in_fragment
In Excel, the log(base2) enrichment ratio of the wildtype sequence was subtracted from the log(2) enrichment ratios for all single mutations.
Assembly: Not applicable
Supplementary files format and content: Excel spreadsheet of log(base2) enrichment ratios for each single amino acid substitution.  Also includes the total reads for each mutation in the naive plasmid library.

extract_protocol:
Total RNA was extracted from FACS sorted cells using a GeneJet kit (Thermo Scientific), and cDNA was synthesized using high fidelity Accuscript (Agilent) primed from one of two gene-specific primers.  The EFNB2 cDNA was PCR amplified as two separate fragments that together fully cover the diversified regions of EFNB2 present in the SSM library.  The PCR fragments were amplified for Illumina sequencing in two rounds.  In the first round, primer overhangs added sequences for annealing to the Illumina sequencing primers. In the second round, primer overhangs added unique barcodes and Illumina adaptors.  To determine the frequencies of mutations in the naive library, the library plasmid DNA was also used as a template for PCR amplification and Illumina sequencing.
Primer used for reverse transcription with Accuscript for all fragments: pCEP4_EBV_Reverse (GTGGTTTGTCCAAACTCATC)
Primer pair for 1st round of PCR (Fragment A): MiSeq_CCR5_myctag_F (TCTTTCCCTACACGACGCTCTTCCGATCTCGAGCAAAAGCTGATCTCAG) and Illumina_EFNB2_295_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTTCAGAAGAGGCGTATTCTC)
Primer pair for 1st round of PCR (Fragment B): Illumina_EFNB2_271_F (TCTTTCCCTACACGACGCTCTTCCGATCTGCGGATCGCTGTACTATCAA) and Illumina_EFNB2_525_R (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATTCCGCGTTGATCCCGCT)
Primer pair for 2nd round of PCR (added I5 and I7 Illumina adaptors): MiSeq_Start_Adaptamer (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT) and MiSeq_Index_Adaptamer (CAAGCAGAAGACGGCATACGAGAT-6nt-barcode-GTGACTGGAGTTCAGACGTGTGCTCTTC)
Amplicons were deep sequenced on a NovaSeq 6000 using a 2x250 nt paired end protocol.

treatment_protocol: Expi293F cells were transfected using expifectamine (ThermoFisher) with a pCEP4 episomal plasmid-based library encoding variants of myc-tagged ENFB2 diluted with a 1500-fold excess of carrier DNA; these transfection conditions ensure that most cells express no more than a single EFNB2 sequence variant, thereby linking genotype to phenotype.  Cells were harvested 24 h post-transfection and co-incubated with anti-myc Alexa 647 (1/250 dilution) and 150 nM sEphB2-Fc.  Cells were washed, incubated with cleared medium from cells secreting NiV-G-sfGFP (1/5 dilution), washed again, and sorted by FACS.  Gates were drawn for the main population of viable cells using forward and side scatter properties and exclusion of the fluorescent viability dye propidium iodide.  The EFNB2-positive cells based on Alexa 647 fluorescence were further gated to collect the top 10% and bottom 15% of cells for bound NiV-G-sfGFP relative to EFNB2 expression.

value: Bottom 15% of cells for bound NiV-G-sfGFP relative to surface EFNB2 expression


title: Participant 02, timepoint 3

description: ex vivo whole blood

value: whole blood

value: hr 6


title: PA14_DrsaLDlasR_16_llras_B

source_name: RNASeq of planctonic culture of Pseudomonas aeruginosa UCBPP-PA14 delta rsaL delta lasR pSEVA224:: lasR_long pSEVA644::rsaL_short treated for 2 h with 0.1 mM IPTG  at OD (0.5-0.7)

value: PA14 delta rsaL delta lasR pSEVA224::lasR_long pSEVA644::rsaL_short


title: Participant 02, timepoint 1

description: ex vivo whole blood

value: Plasmodium vivax infection

value: hr 0


title: OCT4-GFP positive germ cells, GR flox control, biological rep 1

description:
CFF0001
Tag-Seq_featureCounts_aggregate_female.csv
Female-cKO_vs_Female-flox.csv


title: EFNB2 library sorted for low NiV-G binding (Replicate 1; PCR Fragment A  Fragment B)


title: Participant 02, timepoint 4

description: ex vivo whole blood

value: Plasmodium vivax infected

value: hr 9


title: OCT4-GFP positive germ cells, GR conditional knockout, biological rep 4

description:
CFC0004
Tag-Seq_featureCounts_aggregate_female.csv
Female-cKO_vs_Female-flox.csv


title: EFNB2 library sorted for high NiV-G binding (Replicate 1; PCR Fragment A Fragment B)

value: Top 10% of cells for bound NiV-G-sfGFP relative to surface EFNB2 expression


title: Telencephalon, not building, biol rep 5

city: Atlanta

first: Larry

last: Young

state: GA

address: 954 Gatewood Rd NE

department: Department of Psychiatry

institute: Emory University

description: 10x Genomics

data_processing:
Demultiplexing, barcode processing, and gene counting was conducted using Cell Ranger software v3.1.0
Assembly: M_zebra_UMD2a
Supplementary files format and content: Gunzipped tab-separated values files, matrix files, and comma-separated value files

organism: Mchenga conophoros

source_name: Telencephalon

extract_protocol:
Frozen telencephalon tissue samples were incubated on ice for 30 minutes in chilled lysis buffer (10 mM Tris-HCL, 10 mM NaCl, 3 mM MgCl2, 1% Nonidet P40 Substitute, and Nuclease-free H2O) with gentle rotation. Following lysis, HAB medium (with 2% B27 and 0.5 mM Glutamax) was added and tissue was triturated using silanized glass Pasteur pipettes (500 μm internal diameter) to complete tissue dissociation. Samples were centrifuged (600rcf, 5 minutes, 4°C) and resuspended in chilled wash and resuspension buffer (1X PBS, 2% BSA, 0.2 U/μl RNase Inhibitor). Nuclei suspensions were filtered through 40 μm Flowmi® cell strainers and 30 μm MACS® SmartStrainers to remove debris and large nuclei aggregations. Finally, fluorescence activated cell sorting (FACS) was used to further purify samples. DAPI (1.0 μg/ml) was used as a nuclear stain and viable, intact nuclei singlets were selected and sorted into collection buffer (1X PBS, 2% BSA, 0.2 U/μl RNase Inhibitor) using a BD FACSAriaTM Fusion Cell Sorter.
Libraries were prepared according to manufacturer instructions (Chromium Single Cell 3’ Reagent Kits User Guide v3.1 Chemistry (10X Genomics)). Single nuclei suspensions were loaded onto the 10x Genomics Chromium Controller and partitioned into individual GEMs (gel bead-in emulsion), each containing a single nucleus, a barcoded gel bead and master mix with reverse transcription (RT) reagents. Primers released from each gel bead include a 10X barcode, unique molecular identifier (UMI) and Illumina R1 primer. Partitioned nuclei were lysed and GEMs were incubated to produce full-length, barcoded cDNA. Barcoded cDNA were purified using Silane magnetic beads and amplified via PCR . Amplified cDNA was size-selected with SPRIselect. During final library construction, an Illumina R2 primer sequence, I5 and I7 paired-end construct sequences, and a sample index were added. Quality was assessed using high sensitivity DNA analysis on the Bioanalyzer 2100 system (Agilent) and libraries were quantified using Qubit 2.0 (Invitrogen).
snRNA-seq

value: Telencephalon

tag: behavioral state_before_euthanization

value: not building

tag: age

value: adult (>180 days)


title: OCT4-GFP positive germ cells, GR conditional knockout, biological rep 3

description:
CFC0003
Tag-Seq_featureCounts_aggregate_female.csv
Female-cKO_vs_Female-flox.csv


title: Library of EFNB2 variants (PCR Fragment A  Fragment B)

value: plasmid DNA


title: OCT4-GFP positive germ cells, GR conditional knockout, biological rep 2

description:
CFC0002
Tag-Seq_featureCounts_aggregate_female.csv
Female-cKO_vs_Female-flox.csv

value: ovary


title: PA14_DrsaLDlasR_16_llras_A


title: Telencephalon, not building, biol rep 4

description: 10x Genomics


title: OCT4-GFP positive germ cells, GR conditional knockout, biological rep 1

description:
CFC0001
Tag-Seq_featureCounts_aggregate_female.csv
Female-cKO_vs_Female-flox.csv

value: OCT4-GFP positive germ cells


title: Telencephalon, not building, biol rep 3

description: 10x Genomics


title: Carotid_4

city: Chicago

first: Matthew

middle: J

last: Feinstein

address: 303 E Chicago Ave, Tarry 12-723

institute: Northwestern University

description: 10x Genomics

data_processing:
Sequencing reads were demultiplexed and aligned to the human GRCh38 transcriptome using the CellRanger V3 software (10X Genomics).
Sample matrices were imported into the Seurat v4 R package and combined into a Seurat object. One Seurat object for femoral plaque samples and carotid plaque samples.
Both Seurat objects, cells were filtered for mitochondrial reads <10%, 200 < nCount_RNA < 10 000, and 200 < nFeature_RNA < 10 000 and then to remove mitochondrial (MT-) and ribosomal (RP-) genes.
Each Seurat object was also normalized and scaled and filtered to keep the top variable features (greatest standardized variance; n = 3000) across the datasets. The objects were then merged using the Seurat merge command and integrated using the R package Harmony.
Harmony was used to perform PCA, which is then used to perform unsupervised clustering in the R package, ClusterProfiler.
Assembly: GRCh38
Supplementary files format and content: barcodes.tsv.gz: list of spatial barcodes
Supplementary files format and content: features.tsv.gz: list of gene IDs
Supplementary files format and content: matrix.mtx.gz: gene expression count data in Matrix Market Exchange Format

source_name: human plaque isolated cells

extract_protocol:
Freshly collected femoral and carotid plaque samples were washed thoroughly in Dulbecco’s modified Eagle medium (DMEM), digested at 37°C for 1 hour in: 10 milliliters of DMEM with 10% FBS; type IV collagenase at 1 mg/ml final concentration; and DNase hyaluronidase, collagenase type XI and collagenase type II each at 0.3 mg/ml final concentration. The mixture was filtered consecutively through 70 then 40 micrometer strainers, washed twice in PBS, and centrifuged at 300gfor 8 minutes.
The suspensions were then enriched for live cells and CD45+ cells. Dead cells were removed with a dead cell removal kit. Suspensions were then enriched for immune cells with CD45 positive selection using a CD45+ enrichment kit. CD45+ enriched single cell suspensions of plaque were converted to barcoded scRNA-seq libraries using the Chromium Single Cell 3ʹ Library, Gel Bead, and Chip Kit from 10X Genomics. The Chromium Single Cell 3’ V3.1 Reagent (10x Genomics, PN-1000286) kit was used to prepare scRNA-seq libraries.

tag: plaque source

value: Carotid


title: Femoral_9

description: 10x Genomics

value: Femoral


title: Telencephalon, not building, biol rep 2

description: 10x Genomics


title: endometrial macrophages, IUA+primed MSCs treatment, Primed3

city: Nanjing

first: Simin

last: Yao

address: 321 Zhongshan Road

institute: The Affiliated Drum Tower Hosipital, Medical School, Nanjing University

description: Primed3 from IUA+primed MSCs treatment group

data_processing:
Illumina Casava1.7 software used for basecalling.
We used fastp for quality control of raw reads, filtered low quality data, and obtained clean reads.
Reference genome-based alignment analysis was performed with the use of HISAT2 software.
Based on the HISAT2 alignment results, we reconstructed transcripts using Stringtie and calculated the expression of all genes in each sample using RSEM.
To identify differentially expressed mRNAs across samples or groups, the DESeq2 package (http://www.bioconductor.org/) was used. We identified mRNAs with a fold change ≥2 and a P value <0.05 in a comparison between samples or groups as significant differentially expressed genes.
Assembly: mm10
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample

source_name: endometrium

extract_protocol:
Mice endometrial tissues were cut into small pieces and digested with 0.1% Trypsin (Wisent Inc., Canada) for 8 min and then transferred into Type I Collagenase (0.8mg/mL, Sigma-Aldrich, USA) for 90min at 37°C, 5% CO2, with regular vigorous shaking in a humidified incubator. The released endometrial cells were filtered through 70μm cell strainer (BD Bioscience, USA), centrifuged and re-suspended in 1mL of red blood cell lysis buffer (eBioscience, USA) for 2 min to exclude any remaining red blood cells. Then the mice endometrial cells were re-suspended in PBS and stained with Anti-F4/80 MicroBeads UltraPure (Miltenyi Biotec, Germany) for 15 min at 4℃. The marked cells were washed and resuspended in MACS buffer (Miltenyi Biotec, Germany), and underwent magnetic separation with MS columns according to the manufacturer’s protocol. The final sorted F4/80+ cells were collected for RNA havesting with Trizol reagent.
1 ug of total RNA was used for the construction of sequencing libraries. After enrichment of eukaryotic mRNAs with polyA tails by magnetic beads with Oligo(dT), mRNAs were interrupted with buffer. The first strand of cDNA was synthesized using fragmented mRNA as template and random oligonucleotides as primers in M-MuLV reverse transcriptase system, followed by RNA strand degradation by RNaseH. The second strand of cDNA was synthesized using dNTPs under DNA polymerase I system. The purified double-stranded cDNA was end repaired, an A-tail added, and sequencing adapter ligated. About 200bp cDNA was screened by AMPure XP beads, and underwent PCR amplification. The PCR products were purified by AMPure XP beads again to obtain the library.

value: endometrium

value: macrophages

tag: treatment

value: IUA+primed MSCs treatment


title: Telencephalon, not building, biol rep 1

description: 10x Genomics


title: Telencephalon, building, biol rep 5

description: 10x Genomics

value: building


title: OCT4-GFP positive germ cells, GR flox control, biological rep 2

description:
CFF0002
Tag-Seq_featureCounts_aggregate_female.csv
Female-cKO_vs_Female-flox.csv

tag: developmental stage

value: female


title: EFNB2 library sorted for high NiV-G binding (Replicate 2; PCR Fragment A Fragment B)


title: endometrial macrophages, IUA+primed MSCs treatment, Primed2

description: Primed2 from IUA+primed MSCs treatment group


title: Carotid_3

description: 10x Genomics


title: endometrial macrophages, IUA+primed MSCs treatment, Primed1

description: Primed1 from IUA+primed MSCs treatment group


title: Femoral_7

description: 10x Genomics


title: Telencephalon, building, biol rep 3

description: 10x Genomics


title: endometrial macrophages, IUA+control MSCs treatment, Ctrl3

description: Ctrl3 from IUA+control MSCs treatment group

value: IUA+control MSCs treatment


title: Femoral_6

description: 10x Genomics


title: endometrial macrophages, IUA+control MSCs treatment, Ctrl2

description: Ctrl2 from IUA+control MSCs treatment group


title: Telencephalon, building, biol rep 1

description: 10x Genomics


title: Femoral_4

description: 10x Genomics


title: endometrial macrophages, IUA+control MSCs treatment, Ctrl1

description: Ctrl1 from IUA+control MSCs treatment group


title: endometrial macrophages, IUA modeling, Matrigel3

description: Matrigel3 from IUA group

value: IUA modeling


title: Carotid_1_2

description: 10x Genomics

value: Pooled Carotid


title: Femoral_8

description: 10x Genomics


title: endometrial macrophages, IUA modeling, Matrigel1

description: Matrigel1 from IUA group


title: Femoral_1_2_3

description: 10x Genomics

value: Pooled Femoral


title: endometrial macrophages, IUA modeling, Matrigel2

description: Matrigel2 from IUA group


title: Young_nmrHAS2_M_2

city: Rochester

first: Fathima

last: Athar

state: New York

address: 500 Joseph C Wilson Blvd.

department: Biology

institute: University of Rochester

data_processing:
The Illumina Miseq data analysis was carried out with a workflow employing the Quantitative Insights Into Microbial Ecology (QIIME2) pipeline
The reads were processed the as following: reads were filtered for not matching barcodes; OTU picking and chimera removal was done via matching the sequences to the Silva 111 database, with only one mismatch allowed, and a biom and with clustalw a multiple alignment and phylogenetic tree file was generated. Further outputs were generated via QIIME, such as filtered reads per sample, PD whole tree diversity measurements and the level 1–6 taxonomic distributions with relative abundances. 37,000 reads cutoff was used for all the samples.
Assembly: Silva 111 database
Supplementary files format and content: tab-delimited text files includes filtered reads for each sample
Library strategy: microbiome

source_name: feces

extract_protocol: Fresh fecal samples from 9- and 23-months old animals were obtained in the morning, immediately snap freeze in liquid nitrogen, and stored in -80℃. These samples were used for 16S rRNA gene analysis for microbiota profiling from the V1–V2 region of 16S rRNA genes. DNA extraction was performed using QIAamp DNA Stool Mini Kit (Qiagen) following the manufacture’s instruction. DNA was eluted in 50 µl DNAse free water. Twenty nanograms of DNA were used for the amplification of the 16S rRNA gene with primers 27F-DegS (5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTTYGATYMTGGCTCAG-3’) and 338R I (5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCWGCCTCCCGTAGGAGT-3’) + 338R II (5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCWGCCACCCGTAGGTGT-3’)87 for 25 cycles. Primers have Illumina sequencing index attached; Index forward (5’- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3’) and index reverse (5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3’). The PCR was performed in a total volume of 50 µl containing 1× HF buffer (New England BioLabs), 1 µl dNTP Mix (New England BioLabs), 1 U of Phusion® Hot Start II High-Fidelity DNA polymerase (New England BioLabs), 500 nM of the 27F-DegS primer, 500 nM of an equimolar mix of two reverse primers, 338R I and II. The size of the PCR products (~375 bp) was confirmed by gel electrophoresis using 5 µl of the amplification reaction mixture on a 1% (w/v) agarose gel. The PCR products were purified using QIAquick PCR Purification Kit (Qiagen). Then, the dual indices and Illumina sequencing adapters were attached using the Nextera XT index kit (Illumina) following the manufacture’s instruction. Purified amplicon pools were 250 bp paired end sequenced using Illumina Miseq.

value: feces

value: Young

tag: genotype


title: Young_nmrHAS2_M_4


title: endometrial macrophages, sham, Sham2

description: Sham2 from sham group

value: untreated


title: endometrial macrophages, sham, Sham3

description: Sham3 from sham group


title: Young_nmrHAS2_M_5


title: Young_nmrHAS2_F_5


title: JD9: EE spheroid day 3

city: AUBURN

first: Amit

middle: Kumar

last: Mitra

state: AL

address: HARRISON COLLEGE OF PHARMACY

department: DRUG DISCOVERY AND DEVELOPMENT

institute: AUBURN UNIVERSITY

data_processing:
Partek Genomics Suite
Read count extraction were performed using CPM method (Partek Flow)
Supplementary files format and content: hg38
Supplementary files format and content: tab-delimited text files include raw count values for each Sample

source_name: Prostate

extract_protocol:
High-quality RNA was extracted using QIAshredder and RNeasy kit (Qiagen)
RNA was harvested using Rneasy mini plus kit (Qiagen). 1.3 ug of total RNA was used for the construction of sequencing libraries.
RNAseq libraries were constructed using Illumina TruSeq RNA Sample Preparation kit v2. Libraries were then size-selected to generate inserts of ~200 bp.

value: Prostate

tag: cell line

value: prostate cancer

value: WT

value: METRO


title: JD8: EE (extended exposure) spheroid day 1


title: endometrial macrophages, sham, Sham1

description: Sham1 from sham group


title: JD10: EE spheroid day 7


title: Telencephalon, building, biol rep 4

description: 10x Genomics

value: Telencephalon

value: adult (>180 days)


title: Young_nmrHAS2_M_1


title: Femoral_5

description: 10x Genomics

value: Femoral


title: JD7: CON spheroid day 7

value: Conventoinal


title: JD6: CON spheroid day 3


title: Telencephalon, building, biol rep 2

description: 10x Genomics

value: building


title: Young_nmrHAS2_F_3


title: hUC-MSCs, IL-1β, TNF-a and IFN-γ treated, primed 3

data_processing:
Illumina Casava1.7 software used for basecalling.
We used fastp for quality control of raw reads, filtered low quality data, and obtained clean reads.
Reference genome-based alignment analysis was performed with the use of HISAT2 software.
Based on the HISAT2 alignment results, we reconstructed transcripts using Stringtie and calculated the expression of all genes in each sample using RSEM.
To identify differentially expressed mRNAs across samples or groups, the DESeq2 package (http://www.bioconductor.org/) was used. We identified mRNAs with a fold change ≥2 and a P value <0.05 in a comparison between samples or groups as significant differentially expressed genes.
Assembly: hg18
Supplementary files format and content: tab-delimited text files include FPKM values for each Sample

source_name: hUC-MSCs

growth_protocol: hUC-MSCs were maintained in LG-DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37℃.

extract_protocol:
RNA was harvested using Trizol reagent. 1 ug of total RNA was used for the construction of sequencing libraries.
After enrichment of eukaryotic mRNAs with polyA tails by magnetic beads with Oligo(dT), mRNAs were interrupted with buffer. The first strand of cDNA was synthesized using fragmented mRNA as template and random oligonucleotides as primers in M-MuLV reverse transcriptase system, followed by RNA strand degradation by RNaseH. The second strand of cDNA was synthesized using dNTPs under DNA polymerase I system. The purified double-stranded cDNA was end repaired, an A-tail added, and sequencing adapter ligated. About 200bp cDNA was screened by AMPure XP beads, and underwent PCR amplification. The PCR products were purified by AMPure XP beads again to obtain the library.

treatment_protocol: hUC-MSCs were treated with 10ng/ml IL-1β (R&D system, USA), 20ng/ml TNF-α (R&D system, USA) and 20ng/ml IFN-γ (R&D system, USA) for 24 hours.

value: hUC-MSCs

value: mesenchymal stem cells

value: IL-1beta, TNF-a and IFN-gamma treated


title: Young_nmrHAS2_F_4


title: hUC-MSCs, IL-1β, TNF-a and IFN-γ treated, primed 2


title: JD5: CON (conventional) spheroid day 1


title: hUC-MSCs, IL-1β, TNF-a and IFN-γ treated, primed 1


title: Young_nmrHAS2_F_2


title: JD4: CTRL spheroid day 7

value: Control


title: Young_nmrHAS2_M_3


title: Young_nmrHAS2_F_1


title: Young_CreER_M_5

value: CreER


title: JD2: CTRL spheroid day 1


title: Young_CreER_M_4

value: feces


title: hUC-MSCs, untreated, ctrl 3


title: JD1: CTRL (control) spheroid day 0


title: CH-3

city: Dallas

first: Jiwoong

last: Kim

state: Texas

address: 5323 Harry Hines Blvd.

institute: UT Southwestern Medical Center

data_processing:
Trim Galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) was used for quality and adapter trimming. STAR (v2.7.2b) (Dobin et al., 2013) was employed to align the reads onto the human genome, SAMtools (v1.9) (Li et al., 2009) was employed to sort the alignments, and HTSeq Python package (Anders et al., 2015) was employed to count reads per gene.
Assembly: hg38
Supplementary files format and content: tab-delimited text files include gene read counts for each sample

source_name: culture cells

extract_protocol:
DLD1 colon cancer cells incubated with DMSO, TCDD, or Kyn for 1 h were harvested and RNA was extracted and  subjected to RNA-seq
The qualities of sequencing reads were evaluated using NGS QC Toolkit (v2.3.3) (Patel and Jain, 2012) and high-quality reads were extracted. The human reference genome sequence and gene annotation data hg38 were downloaded from Illumina iGenomes. (https://support.illumina.com/sequencing/sequencing_software/igenome.html). The qualities of RNA-sequencing libraries were estimated by mapping the reads onto human transcript and ribosomal RNA sequences (Ensembl release 89) using Bowtie (v2.3.2) (Langmead and Salzberg, 2012). STAR (v2.5.2b) (Dobin et al., 2013) was used to align the reads onto the human and viral genomes. SAMtools (v1.9) (Li et al., 2009) was used to sort the alignments, and HTSeq Python package was used to count reads per gene (Anders et al., 2015). DESeq2 R Bioconductor package was used to normalize read counts and identify differentially expressed (DE) genes (Gentleman et al., 2004) (Anders and Huber, 2010). KEGG (Kanehisa et al., 2017) pathway data was downloaded using KEGG API (https://www.kegg.jp/kegg/rest/keggapi.html) and gene ontology (GO) data was downloaded from NCBI FTP (ftp://ftp.ncbi.nlm.nih.gov/gene/DATA/gene2go.gz). The enrichment of DE genes to pathways and GOs were calculated by Fisher's exact test in R statistical package.

value: culture cells

value: colon cancer

value: CH


title: Young_CreER_M_2

value: Young


title: JD3: CTRL spheroid day 3

value: Prostate

value: prostate cancer

value: WT


title: hUC-MSCs, untreated, ctrl 2

value: untreated


title: Young_CreER_M_1


title: Young_CreER_M_3


title: CH-1


title: Kyn-3

value: Kyn


title: CH-2


title: Young_CreER_F_4


title: Kyn-2


title: Young_CreER_F_5


title: TMA1_20221115 | 023 | Full ROI

city: Basel

first: Sarp

last: Uzun

address: Schönbeinstrasse 40

department: Pathology

country: Switzerland

institute: University Hospital Basel, Institute of Medical Genetics and Pathology

description:
DSP-1001660004682-C-E11
geo_submission_spatial_transcriptomics_Q3_normalized_dataset

data_processing:
Novaseq-derived FASTQ files for each sample were compiled for each compartment using the bcl2fastq program of Illumina, and then demultiplexed and converted to digital count conversion (DCC) files using the GeoMx DnD pipeline version 2.0.0.16 of Nanostring according to manufacturer’s pipeline. DCC files were imported back into the GeoMx DSP instrument for QC and data analyses using GeoMx DSP analysis suite version 2.4.2.2 (Nanostring).  Probes were checked for outlier status. A probe is removed if the geometric mean of that probe’s counts from all segments divided by the geometric mean of all probe counts representing the target from all segments is less than 0.1 or if the probe is an outlier according to the Grubb’s test in at least 20% of segments.   The counts for all remaining probes for a given target were then collapsed into a single metric by taking the geometric mean of probe counts.   Segments and genes were filtered out with abnormal low signal. Segments and genes with less than 5% of gene or segments detected and below the limit of quantification (LOQ) set at 2 geometric standard deviation above the geometric mean were removed from the study.  Gene counts were normalized to the geometric mean of the 75th percentile across all AOIs to give the upper quartile (Q3) normalization factors for each AOI.
Supplementary files format and content: Excel file containing segment properties, target properties, Q3 normalized target count matrix

source_name: Liver

extract_protocol:
Three FFPE tissue sections of 5-µm from liver biopsy tissue microarray (TMA) with VILI (n=6) and AIH (n=8) were mounted on positively charged histology slides. Sections were incubated at 65 °C for 1 hour. Then, sections were deparaffinized in 3 xylol baths of 5 minutes and rehydrated in ethanol gradient from 100% EtOH 2 baths of 5 minutes, followed by 95% EtOH 5 minutes. Slides were then washed in 1X PBS. Antigen retrieval was carried out with Tris-EDTA pH 9.0 buffer at 100°C for 20 minutes at low pressure. Slides were first immersed into hot water for 10 seconds and then immersed into Tris-EDTA buffer. The cooker vent kept open during the procedure to ensure low pressure and it was allowed to reach 100°C. Slides were then washed with 1X PBS, and incubated in proteinase K containing PBS (1ug/ml) for 15 minutes at 37°C and washed again in 1X PBS. Tissue were post-fixed in 10% neutral-buffered formalin (NBF) for 5 minutes, washed two times 5 minutes in NBF stop buffer (0.1M Tris Base, 0.1M Glycine) and finally one time in 1X PBS. The mix of Whole Transcriptome Atlas probes (WTA, Nanostring) was dropped on each section and covered with HybriSlip Hybridization Covers. Slides were then incubated for hybridization overnight at 37°C in a Hyb EZ II hybridization oven (Advanced cell Diagnostics). The day after, HybriSlip covers were gently removed and 25 minutes stringent washes were performed twice in 50% formamide and 2X saline sodium citrate (SSC) at 37 °C. Tissues were washed for 5 min in 2× SSC, then blocked in Buffer W (Nanostring Technologies) for 30 min at room temperature in a humidity chamber. Next, 500 nM Syto83 and antibodies targeting CD3 (BioRad, clone CD3-12), CD20 (Novus, clone IGEL/773) and Arginase (Cell Signaling, clone D4E3M) in Buffer W were applied to each section for 1 h at room temperature. Slides were washed twice in fresh 2× SSC then loaded on the GeoMx DSP.  Entire slides were imaged at ×20 magnification and morphologic markers were used to select Region Of Interest (ROI) either using circle or organic shapes. Automatic segmentation of ROI based on Arginase+ markers were used to defined Area of Illumination (AOIs). This allowed to separate liver cells (Arginase+) and cells around liver. A total of 59 AOIs were exposed to 385 nm light (UV), releasing the indexing oligonucleotides which were collected with a microcapillary and deposited in a 96-well plate for subsequent processing. The indexing oligonucleotides were dried down overnight and resuspended in 10 μl of DEPC-treated water.
Sequencing libraries were generated by PCR from the photo-released indexing oligos and AOI-specific Illumina adapter sequences, and unique i5 and i7 sample indices were added. Each PCR reaction used 4 μl of indexing oligonucleotides, 4 μl of indexing PCR primers, 2 μl of Nanostring 5X PCR Master Mix. Thermocycling conditions were 37 °C for 30 min, 50 °C for 10 min, 95 °C for 3 min; 18 cycles of 95 °C for 15 s, 65 °C for 1 min, 68 °C for 30 s; and 68 °C for 5 min. PCR reactions were pooled and purified twice using AMPure XP beads (Beckman Coulter, A63881), according to the manufacturer’s protocol. Pooled libraries were single-sequenced at 27 base pairs and with the single-index workflow on an Illumina NovaSeq S4 instrument.

tag: cohort

value: AIH

tag: label

value: Liver

value: DSP Barcode


title: Young_CreER_F_2

tag: tissue


title: TMA1_20221115 | 022 | Liver cells

description:
DSP-1001660004682-C-E10
geo_submission_spatial_transcriptomics_Q3_normalized_dataset


title: TMA1_20221115 | 021 | Liver cells

description:
DSP-1001660004682-C-E09
geo_submission_spatial_transcriptomics_Q3_normalized_dataset


title: TMA1_20221115 | 019 | Liver cells

description:
DSP-1001660004682-C-E07
geo_submission_spatial_transcriptomics_Q3_normalized_dataset

value: VILI


title: hUC-MSCs, untreated, ctrl 1

value: hUC-MSCs

value: mesenchymal stem cells


title: TMA1_20221115 | 020 | Liver cells

description:
DSP-1001660004682-C-E08
geo_submission_spatial_transcriptomics_Q3_normalized_dataset


title: TMA1_20221115 | 024 | Full ROI

description:
DSP-1001660004682-C-E12
geo_submission_spatial_transcriptomics_Q3_normalized_dataset


title: TMA1_20221115 | 018 | Liver cells

description:
DSP-1001660004682-C-E06
geo_submission_spatial_transcriptomics_Q3_normalized_dataset


title: Young_CreER_F_3

value: CreER


title: Young_CreER_F_1

value: feces


title: TCDD-2

value: culture cells

value: TCDD


title: TCDD-3

value: colon cancer


title: Kyn-1


title: SEV 1-4

city: shanghai

first: yanyong

last: cheng

address: 639 Zhizaoju Road

department: Department of Anesthesiology

institute: Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine

description:
Rhesus macaques
nuclear RNA
matrix.mtx/barcodes.tsv/genes.tsv

data_processing:
Basecalls performed using CASAVA version 1.8.
Cell Ranger was utilized to calculate the raw counts and cell whitelist by default parameter.
1. We applied the Seurat [https://satijalab.org/seurat/] package for cell normalization and cell filtering considering the MT percentage, minimum and maximum gene numbers under following criteria:
* MT% < 20%;
* Cell Gene Number < Median Gene Number * 2;
* Cell Gene Number > 200.
PCA and tSNE analysis was used for the single cell to cell relation description. The fastMNN function (k = 5, d = 50, approximate = TRUE, auto.order = TRUE) from R package scran (v1.10.2) was used to apply mutual nearest neighbor method to correct for batch effect among samples. Graphcluster was utilized for cell clustering and based on the marker gene achieved some cluster was combined. Wilcox rank sum test was then used for marker gene analysis.
Genome_build: Mnul10
Supplementary_files_format_and_content: MTX file and TSV files contain counts for each barcodes and genes.

organism: Macaca mulatta

source_name: nucleus

growth_protocol: The tissues samples of amygdala were surgically removed and the frozen tissue was used to intact nucleus isolation. The nucleus was isolated and purified as previously described with some modifications.

extract_protocol:
Single-cell gel beads in emulsion (GEMs) were generated on a GemCode Single Cell Instrument (10x Genomics). Single-cell RNA-seq cDNA libraries were prepared using Chromium™ Single Cell 3' Reagent Kit v3 (10x Genomics) according to manufacturer's protocol.
RNA libraries were generated by 10x Genomics Single Cell 3’ Library Gel Bead Kit V3 and prepared for sequencing using standard Illumina protocols.

treatment_protocol: Rhesus macaques were exposed to about 2.5%-3% sevoflurane inhalation anesthesia on approximately postnatal day 7 and then again 14 and 28 days, for four hour each time.

tag: gender

value: male

value: hippocampus

tag: passage

value: sevoflurane


title: TMA1_20221115 | 017 | Full ROI

description:
DSP-1001660004682-C-E05
geo_submission_spatial_transcriptomics_Q3_normalized_dataset


title: TMA1_20221115 | 016 | Liver cells

description:
DSP-1001660004682-C-E04
geo_submission_spatial_transcriptomics_Q3_normalized_dataset


title: DMSO-3

value: DMSO


title: SEV 1-3


title: Participant 02, timepoint 16

first: Robert

last: Moseley

address: 124 Science Drive

institute: Duke University

description: ex vivo whole blood

data_processing:
Each participant's set of Fastq files were aligned to human and parasite genome reference files using STAR (version 2.7.5c)  and quantified using RSEM (version 1.3.3)
PlasmoDB-50_PvivaxP01.gff was first converted to a .gtf file using the new code scripts created for this study (see Key resources table of the manuscript)
The STAR command used for building the human STAR index was STAR --runThreadN <int> --runMode genomeGenerate --genomeDir <output_directory> --genomeFastaFiles <reference_sequence_file> --sjdbGTFfile <annotation_file>
The command for building the parasite STAR index was STAR --runThreadN <int> --runMode genomeGenerate --genomeSAindexNbases 11 --genomeDir <output_directory> --genomeFastaFiles <reference_sequence_file> --sjdbGTFfile <annotation_file>
The RSEM command, which was run once for each organism, used for building the human and parasite RSEM index was rsem-prepare-reference --num-threads <int> --gtf <annotation_file> <reference_sequence_file> <output_directory>
The STAR command for aligning reads from a single Fastq file to the human genome was STAR --runThreadN <int> --runMode alignReads --genomeDir <human_STAR_index> --readFilesIn <Fastq_file> --quantMode TranscriptomeSAM --outSAMtype BAM Unsorted --readFilesCommand zcat --outFilterType BySJout --outFileNamePrefix <output_directory> --outFilterIntronMotifs RemoveNoncanonical
The STAR command for aligning reads from a single Fastq file to the parasite genome was STAR --runThreadN <int> --runMode alignReads --genomeDir <parasite_STAR_index> --readFilesIn <Fastq_file> --quantMode TranscriptomeSAM --outSAMtype BAM Unsorted --readFilesCommand zcat --outFilterType BySJout --alignIntronMin 10 --alignIntronMax 3000 --outFileNamePrefix <output_directory> --outFilterIntronMotifs RemoveNoncanonical
Reads from a single Fastq file could map to both organisms' genomes, which can result in inaccurate quantification results. To remove the small number of reads that map to both organisms' genomes, we identified reads that were present in both the human- and parasite-associated BAM files generated from aligning a single Fastq file using STAR. These reads were then removed from both BAM files, producing new human- and parasite-associated BAM files that contained reads uniquely mapped to the respective organism. These new BAM files were used in the quantification step.
The RSEM command for quantification of the reads uniquely aligned to the human genome was rsem-calculate-expression -p <int> --bam --strandedness reverse --fragment-length-mean <avg_fragment_length> --fragment-length-sd <fragment_lenght_std_dev> --no-bam-output <filtered_BAM_file> <human_RSEM_index> <output_directory>
The RSEM command for quantification of the reads uniquely aligned to the parasite genome was rsem-calculate-expression -p <int> --strandedness reverse --fragment-length-mean <avg_fragment_length> --fragment-length-sd <fragment_lenght_std_dev> --bam --no-bam-output <filtered_BAM_file> <parasite_RSEM_index> <output_directory>
Alignment and quantification was performed on the Duke Compute Cluster (https://rc.duke.edu/dcc/) at Duke University which, at the time of use, used CentOS 8 as the operating system and SLURM as the scheduler for the entire system.
Genes which appear in the comprehensive gene annotation file but which were not expressed (i.e. 0 TPM units) at any time point by a participant or parasite population were removed.
Within the GENCODE comprehensive gene annotation file used for this study are 44 genes with duplicate copies in the pseudoautosomal regions on the X and Y chromosomes (Table S2 of manuscript). Since each of these 44 pairs contained two copies of the same gene, quantification resulted in 44 pairs of duplicated expression profiles for each study participant. Because each participant is male, we added together the duplicate expression profiles in the 44 pairs and retained only a single copy of the duplicated genes. In this way we ensured that pseudoautosomal genes could not have an outsized influence in downstream analyses (e.g. circadian phase estimates) if these genes were found to be highly periodic and sufficiently highly expressed.
QuIn this study, species-specific Quantile Normalization was independently applied to each participant's samples and each participant's parasite samples. In particular, genes within each sample (participant, organism, and time point) were sorted according to their TPM values, thereby ranking genes from least expressed to most expressed in each organism and time point. The average TPM value of all genes occupying the same rank in these sorted lists was then computed across all timepoints within each participant and organism and the original TPM value of each gene was replaced with the average of all genes occupying it's same rank. Here care was taken to include all equally-ranked genes, including those which are ambiguously ranked because they have identical TPM values within a sample. Finally, each sample was reordered by gene, to realign the now between-sample normalized expression levels.
Sample 18's hour-24 time point sample was accidentally destroyed prior to RNA sequencing. Expression levels for this time point were interpolated following Quantile Normalization using Piecewise Cubic Hermite Interpolating Polynomial (PCHIP) interpolation.
Assembly: human: gencode.GRCh38.primary_assembly.genome.fa ; parasite: PlasmoDB-50_PvivaxP01_Genome.fasta
genome feature file: human: gencode.v36.primary_assembly.annotation.gtf; parasite: PlasmoDB-50_PvivaxP01.gff
Supplementary files format and content: Each Sample, e.g., Sample 02, has a tab-delimited text file containing quantile normalized TPM values for each sampling time point.
Supplementary files format and content: counts_human.tsv and counts_vivax.tsv files contain the expected read counts as determined by RSEM for each gene.

extract_protocol:
Frozen cell pellets were used for RNA extraction using the Agencourt RNAdvance Blood RNA purification kit and associated protocols. Samples were cleaned before sequencing with Zymo Research OneStepTM PCR Inhibitor Removal Kit.
Libraries were constructed using a kit specific for blood that depleted both ribosomal and globin RNAs.

value: whole blood

tag: disease state

value: Plasmodium vivax infected

tag: time

value: hr 45


title: Old_nmrHAS2_M_5

value: Old


title: SEV 1-2


title: DMSO-2

tag: cell type


title: TCDD-1


title: Old_nmrHAS2_M_4


title: TMA1_20221115 | 015 | Liver cells

description:
DSP-1001660004682-C-E03
geo_submission_spatial_transcriptomics_Q3_normalized_dataset

value: Liver

value: DSP Barcode


title: TMA1_20221115 | 014 | Full ROI

description:
DSP-1001660004682-C-E02
geo_submission_spatial_transcriptomics_Q3_normalized_dataset

value: VILI


title: Old_nmrHAS2_M_3


title: Old_nmrHAS2_M_2


title: Old_nmrHAS2_M_1


title: Ctrl 1-4

value: control


title: Blood_Platelets_TR5161-NSCLC-MAAS

data_processing:
Single-end 100 bp reads were subjected to pre-aligment 5'-end quality trimming and sequence adapter clipping using Trimmomatic.
Cleaned-up reads were mapped to the hg19 reference genome using STAR (version 2.3.0), using splice aware alignments and allowing for 10 mismatches.
Intron-spanning read summarization was performed using a samtools-based filtering step and HTSeq (version 0.6.1) using union-mode, unstranded reads and minimal mapping quality of 35, all guided by the Ensembl gene annotation version 75.
Assembly: hg19
Supplementary files format and content: Samples in the Rdata-file were processed according to the described protocol (after filtering for low-abundent RNAs).

source_name: Thrombocytes

extract_protocol:
Blood platelets were isolated from whole blood in purple-cap BD Vacutainers containing the EDTA anti-coagulant. The cells and aggregates were removed by centrifugation at room temperature for 20 minutes at 120g, resulting in platelet-rich plasma. The platelets were isolated from the platelet-rich plasma by centrifugation at room temperature for 20 minutes at 360g. The platelet pellet was collected in 30 μl RNAlater (Life Technologies), incubated overnight at 4°C and frozen at -80°C for further use. Frozen platelets were thawed on ice and total RNA was isolated using the mirVana RNA isolation kit (Life Technologies) according to the manufacturers’ protocol. Complementary purification of small RNAs was included in the isolation procedure by addition of miRNA homogenate (Life Technologies). Total RNA was dissolved in 30 μl Elution Buffer (Life Technologies) and RNA quality and quantity was measured using Bioanalyzer 2100 with RNA 6000 Picochip (Agilent).
100-500 pg of platelet total RNA diluted in nuclease free H2O was subjected to cDNA synthesis and amplification using the SMARTer Ultra Low RNA Kit for Illumina Sequencing v3 (Clontech, cat. nr. 634853) according to the manufacturers’ protocol. Conversion and efficient amplification of cDNA was quality-controlled using the Bioanalyzer 2100 with DNA High Sensitivity chip (Agilent). Samples with detectable fragments in the 300-7500 bp region were selected for further processing and Covaris shearing by sonication (Covaris Inc). Sample preparation for Illumina sequencing was performed using the Truseq DNA Sample Preparation Kit (Illumina, cat nr. FC-121-2001) or Truseq Nano DNA Sample Preparation Kit (Illumina, cat nr. FC-121-4001). Sample quality and quantity was measured using the DNA 7500 chip or DNA High Sensitivity chip (Agilent). High-quality samples with product sizes between 300-500 bp were pooled in equimolar concentrations and submitted for 100 bp Single Read sequencing on the Hiseq 2500 or 4000 platform (Illumina).

value: Thrombocytes

tag: cancer stage

tag: patient group

value: NSCLC


title: TMA1_20221115 | 013 | Full ROI

description:
DSP-1001660004682-C-E01
geo_submission_spatial_transcriptomics_Q3_normalized_dataset

tag: molecule subtype


title: DMSO-1

value: culture cells


title: Ctrl 1-3


title: TMA1_20221115 | 012 | Liver cells

description:
DSP-1001660004682-C-D12
geo_submission_spatial_transcriptomics_Q3_normalized_dataset


title: Old_nmrHAS2_F_5

value: feces

tag: age


title: TMA1_20221115 | 011 | Liver cells

description:
DSP-1001660004682-C-D11
geo_submission_spatial_transcriptomics_Q3_normalized_dataset


title: Ctrl 1-2


title: Blood_Platelets_TR5128-HD-MAAS

value: Control


title: Blood_Platelets_TR5159-NSCLC-MAAS


title: TMA1_20221115 | 010 | Full ROI

description:
DSP-1001660004682-C-D10
geo_submission_spatial_transcriptomics_Q3_normalized_dataset


title: Ctrl 1-1

value: male


title: Blood_Platelets_TR5127-HD-MAAS


title: TMA1_20221115 | 009 | Liver cells

description:
DSP-1001660004682-C-D09
geo_submission_spatial_transcriptomics_Q3_normalized_dataset


title: Blood_Platelets_TR5126-HD-MAAS


title: Old_nmrHAS2_F_1


title: E15.5 ovary, GR homozygous deletion

description: Illumina 10X Single Cell 3' v3

data_processing:
All demultiplexing, barcode processing, feature counting, and aggregation were performed using Cell Ranger.
All downstream data processing was performed using Seurat v 3.2.3
Assembly: mm10
Supplementary files format and content: Tab-separated barcode files and feature files, as well as matrix files.

source_name: E15.5 ovary, enriched for OCT4-GFP germ cells

extract_protocol:
Fetal ovaries were dissected into ice cold 0.4% BSA in PBS, washed once, transferred to 0.25% Trypsin-EDTA and incubated in a 37°C water bath for 30 minutes, with gentle pipetting every 10-15 minutes to facilitate the dissociation. After 30 minutes, DNase I (1 mg / mL) was added at a 1:10 dilution, and samples were incubated another 10 minutes at 37°C. Samples were pipetted to ensure complete digestion, and then an equal volume of ice-cold FBS was added to inactivate trypsin. To prepare for FACS sorting, Sytox Blue viability dye was added to samples at 1:1000 dilution, and then samples were filtered through a 35 μm filter into FACS tubes. Samples were sorted to a BD FACSAria II, mixed at a 60:40 ratio of germ cells to somatic cells, and then submitted for single cell capture using the 10X Chromium platform.
Libraries were prepared using the Illumina 10X Single Cell 3' v3 kit, as per the manufacturer's instructions.

value: ovary

value: OCT4-GFP germ cells

value: GR deletion

tag: Sex

value: female

tag: mouse strain

value: Tg(Pou5f1-EGFP)2Mnn Nr3c1tm1.1Jda


title: SEV 1-1

value: hippocampus

tag: treatment

value: sevoflurane


title: TMA1_20221115 | 007 | Full ROI

description:
DSP-1001660004682-C-D07
geo_submission_spatial_transcriptomics_Q3_normalized_dataset

value: AIH

value: Liver


title: E15.5 ovary, GR wildtype

description: Illumina 10X Single Cell 3' v3

tag: developmental stage

tag: genotype

value: Wildtype


title: Old_nmrHAS2_F_3

value: Old


title: Old_CreER_M_5

value: CreER


title: TMA1_20221115 | 008 | Liver cells

description:
DSP-1001660004682-C-D08
geo_submission_spatial_transcriptomics_Q3_normalized_dataset

tag: cohort

value: DSP Barcode


title: Old_nmrHAS2_F_2


title: Blood_Platelets_TR5124-HD-MAAS

value: Thrombocytes


title: OCT4-GFP negative somatic cells, saline control, biological rep 3, male

description:
SOMAsal3
gene_count_male.csv
gene_fpkm_male.csv
DGE_SOMA_DEXvsSAL_male_ALL.csv

data_processing:
Raw fastq reads were processed to remove adapter sequences, reads containing poly-N, and low quality reads
Reads were aligned to mm10 reference genome using Hisat2 v2.0.5.
Counts of reads mapping to each gene were determined using featureCounts v1.5.0-p3, followed by calculation of FPKM values.
Pairwise differential expression analysis was performed using DESeq2 v1.20.0. P-values were adjusted using Benjamini and Hochber FDR.
Assembly: mm10
Supplementary files format and content: CSV file containing raw counts for each sample
Supplementary files format and content: CSV file containing FPKM normalized counts for each sample
Supplementary files format and content: CSV file containing pairwise differential gene expression output from DESeq2

source_name: OCT4-GFP negative somatic cells sorted from PN1 testis

extract_protocol:
RNA was harvested using RNeasy Micro Kit (QIAgen)
mRNA was purified from total RNA using poly-dT beads, fragmented, and then used for first strand cDNA sythesis with random hexamer primers (followed by second strand cDNA synthesis). Fragments were end repaired, A-tailed, ligated to sequencing adapters, size selected, PCR amplified, and libraries purified. Libraries were quantified on Qubit and QC'ed on Agilent Bioanalyzer for quality and size distribution.

treatment_protocol: Pregnant dams were dosed by IP injection at E17.5 and E18.5 with either 10 μg dex (Sigma) per gram of body weight, or with an equal volume of saline control. After birth at PN0, pups were injected via subcutnaeous injection with the same dose of dex (or saline), scaled by body weight. Testes were collected at PN1, and germ cells FACS sorted from somatic cells using OCT4-GFP transgene.

value: testis

value: OCT4-GFP negative somatic cells

value: saline


title: Blood_Platelets_TR5125-HD-MAAS

value: Control


title: Old_CreER_M_4

value: feces


title: Old_nmrHAS2_F_4


title: Old_CreER_M_3


title: TMA1_20221115 | 004 | Liver cells

description:
DSP-1001660004682-C-D04
geo_submission_spatial_transcriptomics_Q3_normalized_dataset

tag: label


title: TMA1_20221115 | 005 | Liver cells

description:
DSP-1001660004682-C-D05
geo_submission_spatial_transcriptomics_Q3_normalized_dataset

tag: tissue


title: Blood_Platelets_TR5123-HD-MAAS


title: Old_CreER_M_2


title: OCT4-GFP negative somatic cells, saline control, biological rep 1, male

description:
SOMAsal1
gene_count_male.csv
gene_fpkm_male.csv
DGE_SOMA_DEXvsSAL_male_ALL.csv

value: male


title: OCT4-GFP negative somatic cells, dexamethasone treated, biological rep 3, male

description:
SOMAdex3
gene_count_male.csv
gene_fpkm_male.csv
DGE_SOMA_DEXvsSAL_male_ALL.csv

value: dexamethasone


title: Blood_Platelets_TR5121-HD-MAAS


title: Old_CreER_M_1


title: OCT4-GFP negative somatic cells, dexamethasone treated, biological rep 2, male

description:
SOMAdex2
gene_count_male.csv
gene_fpkm_male.csv
DGE_SOMA_DEXvsSAL_male_ALL.csv


title: Blood_Platelets_TR5120-HD-MAAS


title: TMA1_20221115 | 002 | Full ROI

description:
DSP-1001660004682-C-D02
geo_submission_spatial_transcriptomics_Q3_normalized_dataset


title: Old_CreER_F_4


title: OCT4-GFP negative somatic cells, dexamethasone treated, biological rep 1, male

description:
SOMAdex1
gene_count_male.csv
gene_fpkm_male.csv
DGE_SOMA_DEXvsSAL_male_ALL.csv


title: TMA1_20221115 | 001 | Full ROI

description:
DSP-1001660004682-C-D01
geo_submission_spatial_transcriptomics_Q3_normalized_dataset


title: Old_CreER_F_3


title: Blood_Platelets_TR5119-HD-MAAS


title: TMA1_20221115 | 006 | Liver cells

description:
DSP-1001660004682-C-D06
geo_submission_spatial_transcriptomics_Q3_normalized_dataset


title: OCT4-GFP positive germ cells, saline control, biological rep 2, male

description:
GERMsal2
gene_count_male.csv
gene_fpkm_male.csv
DGE_GERM_DEXvsSAL_male_ALL.csv

source_name: OCT4-GFP positive germ cells sorted from PN1 testis

value: OCT4-GFP positive germ cells


title: OCT4-GFP positive germ cells, saline control, biological rep 3, male

description:
GERMsal3
gene_count_male.csv
gene_fpkm_male.csv
DGE_GERM_DEXvsSAL_male_ALL.csv


title: Old_CreER_F_2


title: TMA1_20221116 | 010 | Liver cells

description:
DSP-1001660004682-C-C12
geo_submission_spatial_transcriptomics_Q3_normalized_dataset

value: VILI


title: OCT4-GFP negative somatic cells, saline control, biological rep 2, male

description:
SOMAsal2
gene_count_male.csv
gene_fpkm_male.csv
DGE_SOMA_DEXvsSAL_male_ALL.csv


title: OCT4-GFP positive germ cells, saline control, biological rep 1, male

description:
GERMsal1
gene_count_male.csv
gene_fpkm_male.csv
DGE_GERM_DEXvsSAL_male_ALL.csv


title: Blood_Platelets_TR5117-HD-MAAS


title: Blood_Platelets_TR5116-HD-MAAS


title: Blood_Platelets_TR5122-HD-MAAS


title: TMA1_20221115 | 003 | Liver cells

description:
DSP-1001660004682-C-D03
geo_submission_spatial_transcriptomics_Q3_normalized_dataset


title: TMA1_20221116 | 008 | Liver cells

description:
DSP-1001660004682-C-C10
geo_submission_spatial_transcriptomics_Q3_normalized_dataset


title: Old_CreER_F_1


title: OCT4-GFP positive germ cells, dexamethasone treated, biological rep 3, male

description:
GERMdex3
gene_count_male.csv
gene_fpkm_male.csv
DGE_GERM_DEXvsSAL_male_ALL.csv


title: OCT4-GFP positive germ cells, dexamethasone treated, biological rep 2, male

description:
GERMdex2
gene_count_male.csv
gene_fpkm_male.csv
DGE_GERM_DEXvsSAL_male_ALL.csv


title: HDR Pool 3 GL2 - Rep 4 gfp -

city: Columbus

first: Jeffrey

last: Parvin

state: OH

address: 460 West 12th Ave

department: Department of Biomedical Informatics

institute: The Ohio State University

data_processing:
For pool 3, the FASTQ files containing barcode variants and a barcode map were processed using Enrich2 software. Enrich2 calculates the functional score for each variant from its abundance in the GFP-positive and GFP-negative pools. The functional score is obtained by normalizing the slope of the line connecting the control and experimental conditions for each pool by the wild-type adjusted by log ratio. The functional score was reported in log2 for each variant tested with a value of 0 representing normal wild-type function.
The process of interpreting variant scores involved two steps. First, two subpopulations of variants containing synonymous and nonsense changes were identified and analyzed in a scatter plot to display the relationship between the variant read count and their functional scores for each subpopulation. Under control conditions, the endogenous BRCA1 present in every cell functioned normally and was used as a baseline for the DNA damage repair assay. Thus, it was anticipated that all variant scores in this condition would cluster around 0 on the scatter plot. The minimum-read count threshold was determined to be the highest point where the results for any variant deviated from this functionally normal value. To confirm this threshold, using the scatter plot from the conditions in which the endogenous BRCA1 was depleted by transfecting siBRCA1-3’UTR, nonsense variants were expected to exhibit negative functional scores indicative of loss of function, and the read-count threshold would be higher than any nonsense mutants that scored as functionally normal. If the read count values from the conditions were different, we selected the higher threshold. Data with lower read counts than this empirically defined value were discarded. To further refine the data, the mean variance across four replicates was calculated, and variants with variance greater than 1 were removed from the analysis. These two filters removed variants with low frequency of read counts and high variability of results. The remaining variants have high frequency of datapoints that were relatively consistent in score, and we used these to evaluate functional effects of the amino acid change for each variant.
After applying the minimum reads-count threshold, a histogram was generated to visualize the distribution of the variant library (Figure S1, bottom right). Three subpopulations, missense, nonsense, and synonymous, were analyzed separately for each condition. To determine the functional score thresholds, the population of missense variants in the control siRNA experimental condition was modeled as a Gaussian distribution, and the lowest 1% of values were selected as the threshold for loss of function. Similarly, using the distribution plot for the BRCA1 siRNA condition, the population of nonsense variants were modeled as a Gaussian distribution and the highest 1% was defined as functionally normal. Only variants that were observed in at least three replicates were included in the analysis.
Assembly: genome_build.txt
Supplementary files format and content: .h5 files with counts and scores. Columns names are descriptive, _- denotes noGFP, _+ denotes GFP samples.

source_name: HeLa

growth_protocol: DMEM supplemented with 10% fetal bovine serum and hygromycin B at 550 ¬µg/mL.

extract_protocol:
Inverse PCR reactions of the plasmid encoding the BRCA1 cDNA generate full length product, but one of the codons in the PCR oligonucleotide contains NNK (where N = A, C, G, T and K = G, T) in place of the terminal codon encoded by the oligonucleotide. For the 97 codons analyzed in this study, 97 different inverted oligonucleotide pairs were separately prepared and subjected to PCR. The PCR reactions were then pooled and circularized by the action of DNA ligase. An oligonucleotide containing a 16 bp degenerate oligonucleotide barcode was inserted upstream of the BRCA1 coding sequence, and long-read sequencing using a PacBio Sequel paired each barcode with a different BRCA1 variant. The plasmid library for pools 3 was transfected along with the FLP-In recombinase into a HeLa derived cell line that contained in its genome a single FLP-In Recombinase Target (FRT) sequence  and a single DNA sequence for measuring homology directed repair (HDR) of DNA double-strand breaks.
The HDR assay is initiated by transfecting a plasmid encoding the I-SceI endonuclease, which generates a DNA cut in one of the defective GFP sequences, and if the cell is competent for HDR, recombination repairs the defective GFP coding sequence, and the cell becomes GFP-positive. We performed this HDR assay under two conditions. In the first condition, we have transfected a control siRNA, and the cells contain endogenously expressed wild-type BRCA1. In the second condition, we have transfected an siRNA targeting the 3’-UTR of the endogenously expressed BRCA1 mRNA, and the cell is then dependent on the BRCA1 variant expressed from the FRT site. GFP-positive cells were separated from GFP-negative cells using flow sorting, and the barcode in the genome of the cells was isolated by PCR and analyzed by short read sequencing. The frequency of the barcode in the GFP-positive cells (functional for HDR) is compared under the conditions of depletion of the endogenous BRCA1 protein using the siRNA targeting the BRCA1 3’-UTR to the condition of the control siRNA. From the abundances of the barcodes in the various samples, we calculated a functional score using the program Enrich2.

treatment_protocol: HDR reporter assays and FACS sorts were performed for the BRCA1 variant libraries in quadruplicate. A confluent 10 cm plate of HeLa BRCA1 variant cell line was trypsinized and resuspended in 10 mL of culture media. 65 ml of the suspension was plated in each of 48 wells across two 24-well tissue culture plates. 24 hours later, each well was transfected with 30 pmol of siRNA and 1.5 ml Oligofectamine (ThermoFisher). Oligofectamine was diluted with 6 ml Opti-MEM and siRNA was diluted with 25 ml Opti-MEM for 5 minutes. The dilutions were then combined and incubated for an additional 30 minutes. The transfection mixture was then applied directly to the cells. 24 hours later, the cells were trypsinized and transferred to four 6-well tissue culture plates, then incubated for another 24 hours. Each well was then transfected with 50 pmol of siRNA, 3 mg pCBASceI (for I-SceI expression), and 3 ml Lipofectamine 2000. Plasmid and siRNA were diluted in 125 ml Opti-MEM and Lipofectamine was diluted in 125 ml Opti-MEM, then incubated for 5 minutes. The dilutions were combined and incubated for an additional 20 minutes. The transfection mixture was then applied directly to cells. Four to six hours later, the culture media was replaced with fresh media. BRCA1 3‚ÄôUTR siRNA, BRCA1 coding sequence siRNA and control siRNA were used in both rounds of transfection. After 24 hours, one well of cells treated with each condition was analyzed for GFP expression using flow cytometry to confirm transfection efficiency. If cells treated with control siRNA and 3‚ÄôUTR siRNA fell within 7-9% and 4-7% GFP+ cells respectively, and cells treated with BRCA1 coding sequence siRNA were 1-2% GFP+, the experiment would proceed. 72 hours post-transfection, the cells were pooled according to treatment and sorted using fluorescent activated cell sorting (FACS) using an Aria IIu instrument. Cells were resuspended and pooled in filter-sterilized sorting buffer containing 1X Phosphate Buffered Saline (Ca2+/Mg2+¬†free), 5 mM EDTA, 25 mM HEPES pH 7.0, and 1% heat-inactivated fetal bovine serum dialyzed against Ca2+/Mg2+¬†PBS. A minimum of 500,000 GFP+ cells and a maximum of 2 million GFP- cells were collected per pool.

tag: cell line

value: HeLa

tag: condition

value: Control group

value: GL2 siRNA


title: HDR Pool 3 GL2 - Rep 3 gfp -


title: TMA1_20221116 | 007 | Liver cells

description:
DSP-1001660004682-C-C09
geo_submission_spatial_transcriptomics_Q3_normalized_dataset

value: Liver


title: Blood_Platelets_TR5118-HD-MAAS


title: OCT4-GFP positive germ cells, dexamethasone treated, biological rep 1, male

description:
GERMdex1
gene_count_male.csv
gene_fpkm_male.csv
DGE_GERM_DEXvsSAL_male_ALL.csv


title: TMA1_20221116 | 005 | Full ROI

description:
DSP-1001660004682-C-C07
geo_submission_spatial_transcriptomics_Q3_normalized_dataset