title: LVAD127


title: LVAD125

status: Public on Mar 11 2021

type: SRA

city: Stanford

first: Mark

middle: Emile

last: Pepin

state: CA

address: 870 Quarry Road

department: Cardiovascular Medicine

country: USA

institute: Stanford University

library_source: transcriptomic

data_processing:
Illumina bcl2fastq version 1.8.4 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence using TrimGalore version 0.4.0 with parameters: --paired, --trim1
Trimmed reads were then aligned with TopHat version 2.0.12 (Bowtie2 version 2.2.3) to UCSC hg19 reference genome with parameters: -r 150, --library-type fr-firststrand, --keep-fasta-order, --transcriptome-index, --rg-id, --rg-sample, --rg-library, --rg-platform-unit, --rg-platform, -o
Cufflinks version 2.2.1 estimated transcript abundances with parameters: --library-type fr-firststrand, -o, -g, -L, -b, -u
Cuffmerge, Cuffquant, Cuffdiff, and Cuffquant were then run to get to final differential expression results
Genome_build: hg19

molecule: total RNA

organism: Homo sapiens

source_name: RNA from cardiac left ventricle

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

treatment_protocol: Human Samples taken from left ventricle of patients with end-stage heart failure during Ventricular Assist Device implantation.

tag: race

value: Caucasian American

tag: age

tag: hypertension

tag: tissue

value: Apical left ventricle


title: sd1-PASCs-2

city: Nanjing

first: Chuanxi

last: Yang

state: China

address: 300 Guangzhou Road, 210029, Nanjing, PR China

department: Department of Cardiology

country: China

institute: School of Medicine, Southeast University

data_processing:
Tophat 2 software used for basecalling.
expected number of Reads Per Kilobase of transcript sequence per Millions base  pairs sequenced (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
Genome_build: rat(rn5)
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

organism: Rattus norvegicus

source_name: perirenal adipose

growth_protocol: Rats were anesthetized with 2% isoflurane to induce sacrifices, soaked in 75% ethanol for whole body disinfection, removed from alcohol, and placed on a sterile ultra clean table. The limbs were fixed, SAT was obtained from the groin. Perirenal and epididymal adipose tissue were obtained from kidney and testis, and the tissue was rinsed in pre-cooled PBS. The washed adipose tissue was transferred into the cell platform, muscle and lymph tissue were removed, tissue was cut into 1–2 mm pieces, and collagenase type I was added. After digestion, all the suspensions collected were filtered through a nylon filter (Dutscher, 100 µm), centrifuged at 1,000 rpm for 5 minutes, and the supernatant was discarded. DMEM / F12 medium containing 10% fetal bovine serum (FBS) was added to the sample. Then the suspension was evenly spread in culture dishes and cultured into a 37°C incubator for 2 hours. The upper medium was discarded, high glucose medium containing 10% FBS was added, and SASCs, PASCs, and EASCs were obtained.

extract_protocol:
P3 SASCs, PASCs, and EASCs were cultured in 12 well plates with six replicates in each group. Each well was ground with 1 mL Trizol solution, placed at room temperature for 5 minutes, and then aspirated. RNA was separated using chloroform (mixture: chloroform = 5:1) and precipitated by isopropanol and absolute ethanol. Finally, RNA was precipitated with DEPC water, and then stored at -80°C for subsequent transcriptome sequencing (Guangzhou Ruibo company，guangzhou, China).
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: strain

value: Sprague Dawley

tag: cell type

value: perirenal adipose-derived mesenchymal stem cells


title: LVAD119

value: Eastern African


title: LVAD111

value: African American


title: pregnant (Days 12-13) vs. cyclic (Days 12-13 of the estrous cycle) 1_2

type: RNA

city: Olsztyn

first: Agata

last: Zmijewska

address: Oczapowski 1a

department: Demartment of Animal Anatomy and Physiology

country: Poland

institute: Univeristy of Warmia and Mazury

hyb_protocol: Two differentially labelled samples (from pregnant and cyclic gilts) were placed on each array (one slide, n = 4) in a balanced block design with dye swaps. Hybridization was carried out in an Agilent hybridization oven for 17 h at 65°C. After the hybridization process, the microarrays were washed.

scan_protocol: Microarrays were scanned at 5 μm resolution with an Agilent’s High-Resolution C Microarray Scanner according to the manufacturers' protocol.

data_processing: The TIFF image, generated after the scanning process, was used for: data extraction and detailed analysis, background subtraction from features, and dye normalizations (linear and LOWESS); in the Feature Extraction Software. Gene expression data were analysed in GeneSpring GX 12 software (Agilent Technologies, USA) to identify the genes that were differentially expressed in pregnant and cyclic pigs.

organism: Sus scrofa domesticus

source_name: Anterior pituitary of pregnant pigs

label_protocol: The RNA isolated from the pituitary of pregnant and cyclic females were labelled with Cyanine-3 (Cy-3) and Cyanine-5 (Cy-5), respectively. Quantity and purity of obtained cRNA were measured with NanoQuant plates on the Infinite M200 Pro plate reader. 825 ng of Cy-3 and 825 ng of Cy-5-labeled cRNA were mixed together (for each microarray). According to the microarray manufacturer’s protocol, cRNA was fragmented and mixed with hybridization buffer.

growth_protocol: Eight mature gilts (Large White x Polish Landrace, weighing  ~110 kg) were used in the study (n = 8). All individuals were descended from private breeding farm. Day 0 was a day indicating the beginning of estrus. Cyclic animals (n=4) were sacrificed on days 12-13.  Days of the estrous cycle were confirmed on the morphology of ovaries. Gilts assigned to the early pregnancy group (n = 4) were naturally bred twice on the first and the second days of the estrous  and sacrificed on days 12-13of pregnancy. Pregnancy was confirmed by the presence of conceptuses. Animals were slaughtered by qualified abattoir worker.

extract_protocol: Total RNA was isolated from porcine pituitaries using the RNeasy Mini Kit (Qiagen, USA), according to manufacture procedure.RNA purity and quantity were measured spectrophotometrically. RNA quality was checked using RNA 6000 Nano Assay Kit on an Agilent 2100 Bioanalyzer. RNA integrity number (RIN) score ≥ 8. The obtained nucleic acid was used for microarray analysis and quantitative real-time PCR (qPCR) experiments. RNAs were stored at −80 °C until the microarray analysis.

tag: reproductive status

value: Early pregnancy

value: Large White x Polish Landrace

tag: days

value: days 12 to 13

source_name: Anterior pituitary of cyclic pigs

value: the estrous cycle


title: LVAD122


title: LVAD121


title: pregnant (Days 12-13) vs. cyclic (Days 12-13 of the estrous cycle) 1_4


title: LVAD153


title: LVAD092


title: genomic DNA from subglottic tissue biopsies [iSGS2]

status: Public on Mar 12 2021

type: genomic

city: Madison

first: Xudong

last: Shi

state: Wisconsin

address: 1111 Highland ave

department: Surgery

institute: University of Wisconsin-Madison

description: iSGS sample

hyb_protocol: bisulphite converted DNA was amplified, fragmented and hybridised to Illumina Infinium Human Methylation27 Beadchip using standard Illumina protocol

scan_protocol: Arrays were imaged using BeadArray Reader using standard recommended Illumina scanner setting

data_processing: Methylation data were preprocessed using the Minfi package within R. Differentially methylated regions were called using the Bumphunter algorithm; differentially methylated CpG sites were called using the dmpFinder function within Minfi on all 865,859 probes.

label: Cy5 and Cy3

molecule: genomic DNA

label_protocol: Standard Illumina Protocol

growth_protocol: Human tissue biopsy.

extract_protocol: Genomic DNA and protein were extracted using a protocol for simultaneous isolation of these biomolecules.Following processing in an AllPrep DNA spin column, DNA (retained in the column), was eluted and reserved.

treatment_protocol: No special treatment

value: Subglottic tissue biopsies


title: genomic DNA from subglottic tissue biopsies [iSGS3]

description: iSGS sample


title: LVAD106


title: LVAD090

value: Asian American


title: LVAD087


title: LVAD084


title: LVAD079


title: LVAD083


title: LVAD075


title: LVAD067


title: LVAD066


title: LVAD054


title: LVAD058


title: LVAD032


title: LVAD013


title: LVAD001


title: P1

city: Yangzhou

first: Wang

last: Li-ping

state: Jiangsu

address: No. 98 West Nantong Road

department: Department of Biobank, Clinical Medical College,

institute: Yangzhou University

data_processing:
Illumina bcl2fastq v1.8.4 software used for basecalling.
Sequenced reads were trimmed fo adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to genome hg19  (February 2009, GRCh37) using HISAT2 v2.1.0 with parameters --dta -x -q --rna-strandness
Reads Per Kilobase of transcript per Million (FPKM) were calculated using StringTie v1.3.3b with default paramters, then differential analysis using R package Ballgown
FEELnc(V 0.1.1) were used to predict LncRNA
Genome_build: GRCh37
Supplementary_files_format_and_content: FPKM

source_name: follicular fluid exosomes

extract_protocol:
The RNA of follicular fluid exosomes was extracted (batch number: 151012397, Qiagen, USA), and the total RNA purity and concentration were measured.
After passing the total RNA quality test, the ribosomal RNA (batch number: 7E010H6, Nanjing Vazyme Co., Ltd.) was removed, and the RNA was fragmented (batch number: 0006358486, Agilent, USA). The cDNA was then synthesized using the fragmented dsRNA as a template, and the final sequencing library was obtained by PCR amplification. After quality inspection, the library was sequenced using an Illumina HiSeq 2500 (Illumina, USA), and the sequencing read length was 2×150 bp (double-ended) (PEl50)

treatment_protocol: Follicular fluids collected during treatment with assisted reproductive technology. After standing for 30 min at room temperature, the fluids were centrifuged at 3000 r/min for 10 min at 4°C. Then, the supernatant was transferred into a clean Amicon Ultra 30 K Centrifugal Device and centrifuged at 10,000 g at 4°C for 30 min. In addition, the supernatant was mixed with 0.2 vol of total exosome isolation reagent. Then, the resulting mixture was incubated at room temperature for 30 min before being centrifuged at 10,000 g at 4°C for 30 min. Finally, the supernatant was discarded, and the pellet was resuspended in phosphate-buffered saline in a 1.5 ml microcentrifuge tube to a final volume equivalent to one fourth of that of the concentrated exosome sample solution.

value: follicular fluid exosomes

tag: Sex

value: female

tag: diagnosis

value: primary infertility accompanied by PCOS (PCOS group)


title: P3


title: N3

value: primary infertility (non-PCOS group)


title: LVAD014


title: LVAD060


title: N1


title: LVAD006


title: P2


title: input_40_lung

city: Amsterdam

first: Tesa

last: Severson

address: 121 Plesmanlaan

department: Molecular Carcinogensis

country: Netherlands

institute: Netherlands Cancer Institute

description: input for Sample 40_lung

library_source: genomic

data_processing:
Raw sequencing data were aligned using BWA v0.5.20 to hg19 and filtered for mapping quality > MQ20 with samtools version 1.8
Duplicate reads were marked with Picard MarkDupes function (version 2.18)
Peaks were called for each sample against it's own input DNA using two peakcallers, MACS2 (v2.1.1) and DFilter (v1.5).For MACS2, we called peaks with the callpeaks function with no model, using a q value threshold cutoff of 0.01 and the fragment size input of the individual sample as determined using phantompeaks (v1.14). Peaks were also called with DFilter using default parameters. We then took the intersect of the peaks called from both peak callers using bedTools intersect (v2.26.0).
Genome_build: hg19
Supplementary_files_format_and_content: peak files in bed format with macs2 output

source_name: Input Sample 40 lung

extract_protocol: Illumina Hi-seq 2500, 65bp single-end reads

tag: disease state

value: metastatic castration resistant prostate cancer

value: lung

tag: chip-antibody


title: input_5_liver

description: input for Sample 5_liver

source_name: Input Sample 5 liver

value: liver


title: H3K27me3_45_Hilar_LN

description: Chromatin IP for H3K27me3 in Sample 45_Hilar_LN

source_name: H3K27me3 ChIP Sample 45 Hilar lymph node

value: Hilar lymph node


title: N2


title: input_45_Hilar_LN

description: input for Sample 45_Hilar_LN

source_name: Input Sample 45 Hilar lymph node


title: H3K27me3_40_lung

description: Chromatin IP for H3K27me3 in Sample 40_lung

source_name: H3K27me3 ChIP Sample 40 lung


title: input_38_Perigastric_LN

description: input for Sample 38_Perigastric_LN

source_name: Input Sample 38 Perigastric lymph node

value: Perigastric lymph node


title: H3K27me3_5_liver

description: Chromatin IP for H3K27me3 in Sample 5_liver

source_name: H3K27me3 ChIP Sample 5 liver


title: H3K27ac_5_liver

description: Chromatin IP for H3K27ac in Sample 5_liver

source_name: H3K27ac ChIP Sample 5 liver


title: H3K27ac_38_Perigastric_LN

description: Chromatin IP for H3K27ac in Sample Perigastric_LN

source_name: H3K27ac ChIP Sample 38 Perigastric lymph node


title: LivD08_2

city: Chongqing

first: Min-Dian

last: Li

address: 30 Gaotanyan Main Street

department: Cardiology

institute: Southwest Hospital, Third Military Medical University (Army Medical University)

data_processing:
illumina Casava 1.8 software use for base calling.
Clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data
Expression levels of genes or transcripts were quantified by featureCounts v1.5.0-p3 (Liao et al., 2014), and represented as transcript per million (TPM).
Genome_build: mm10/GRCm38.p6
Supplementary_files_format_and_content: tab-delimited text files include TPM values for each Sample

molecule: polyA RNA

organism: Mus musculus

source_name: adult female liver

growth_protocol: Special pathogen-free (SPF) mice were purchased from Hunan SJT Laboratory Animal Co. and housed in a SPF barrier facility. C57BL/6J female mice at 7 weeks of age were grouped housed, and entrained to a 12h light:12h dark cycle with normal chow food and water ad libitum for 7 days before assigned to daytime-restricted feeding (DRF) and nighttime-restricted feeding (NRF) groups for additional 7 days.

extract_protocol:
Liver was removed, flash frozen liquid nitrogen, and RNA was harvested using Trizol reagent.
A ribosome-depletion workflow was applied to generate the strand-specific cDNA library as described (Parkhomchuk et al., 2009). Specifically, ribosomal RNAs were removed by Epicenter Ribo-Zero Kit (Epicenter). The remaining RNAs were fragmented (250-300 bp), and reversely transcribed using random hexamers and M-MuLV Reverse Transcriptase (RNase H-). After RNA digestion by RNase H, second strand cDNA synthesis was subsequently performed using dNTP (dTTP replaced by dUTP) and DNA polymerase I, followed by purification, terminal repair, polyadenylation, adapter ligation, size selection, and degradation of second-strand U-contained cDNA. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads (PE155) were generated.

treatment_protocol: Animals were subjected to daytime-restricted feeding (DRF) or nighttime-restricted feeding (NRF) for 7 days

value: 9 weeks

tag: dietary regimen

value: DRF

tag: zeitgeber time in hours


title: H3K27ac_40_lung

description: Chromatin IP for H3K27ac in Sample 40_lung

source_name: H3K27ac ChIP Sample 40 lung


title: FOXA1_5_liver

description: Chromatin IP for FOXA1 in Sample 5_liver

source_name: FOXA1 ChIP Sample 5 liver


title: FOXA1_45_Hilar_LN

description: Chromatin IP for FOXA1 in Sample 45_Hilar_LN

source_name: FOXA1 ChIP Sample 45 Hilar lymph node


title: FOXA1_40_lung

description: Chromatin IP for FOXA1 in Sample 40_lung

source_name: FOXA1 ChIP Sample 40 lung


title: FOXA1_38_Perigastric_LN

description: Chromatin IP for FOXA1 in Sample 38_Perigastric_LN

source_name: FOXA1 ChIP Sample 38 Perigastric lymph node


title: CTCF_5_liver

description: Chromatin IP for CTCF in Sample 5_liver

source_name: CTCF ChIP Sample 5 liver


title: CTCF_38_Perigastric_LN

description: Chromatin IP for CTCF in Sample 38_Perigastric_LN

source_name: CTCF ChIP Sample 38 Perigastric  lymph node

value: Perigastric  lymph node


title: AR_5_liver

description: Chromatin IP for AR in 5_liver

source_name: AR ChIP Sample 5 liver


title: AR_45_Hilar_LN

description: Chromatin IP for AR in Sample 45_Hilar_LN

source_name: AR ChIP Sample 45 Hilar lymph node


title: CTCF_45_Hilar_LN

description: Chromatin IP for CTCF in Sample 45_Hilar_LN

source_name: CTCF ChIP Sample 45 Hilar  lymph node

value: Hilar  lymph node


title: H3K27me3_38_Perigastric_LN

description: Chromatin IP for H3K27me3 in Sample Perigastric_LN

source_name: H3K27me3 ChIP Sample 38 Perigastric lymph node


title: 2mo old normal peripheral lung tissue_rep2

city: Philadelphia

first: Michael

middle: Patrick

last: Morley

state: PA

address: 3400 Civic Center Blvd, Bldg 421

department: Penn Cardiovascular Institute

institute: Perelman School of Medicine at the University of Pennsylvania

data_processing:
The Cell Ranger Single Cell Software Suite was used to perform sample demultiplexing, barcode processing, and single cell 3’ gene counting (http://software.10xgenomics.com/single-cell/overview/welcome). 10bp UMI tags were extracted from Read2.  Genome_build: mm10 or hGRC38
Genome_build: mm10 or hGRC38
Supplementary_files_format_and_content: H5 matrix: contains the UMI counts per feature (gene or peak). BAM:  alignment data. Singlecell: file is the Cellranger Cell annotation file. Fragments: a  BED-like tabular file, where each line represents a unique ATAC-seq fragment captured by the assay.

source_name: 2 mo. old pediatric

extract_protocol: Cellular suspensions were loaded on a GemCode Single Cell Instrument (10x Genomics, Pleasanton, CA) to generate single cell GEMs. Approximately ~1200 to 2800 cells were loaded per channel. Single cell RNA-Seq libraries were prepared using GemCode Single Cell 3’ Gel Bead and Library Kit (now sold as P/N 120230, 120231, 120232, 10x Genomics) following the protocols outlined in Zheng et. al.

tag: genotype

value: normal

value: 2 month


title: 2mo old normal peripheral lung tissue_rep1


title: miR-690oe 4

city: La Jolla

first: Wei

last: Ying

address: 9500 Gilman Dr, Stein Clinical Research Building, Room 225

institute: UC San Deigo

data_processing:
Adapter trimming: Trimmomatic (v.0.38)
polyA removal:FASTP (v.019.4)
QC: RseQC (v.2.5)
DEG: edgeR (v.3.20.9
GO: RSEA (v.3.0)
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include RPKM values

source_name: 3T3-L1 adipocytes with overexpression of miR-690-5p

growth_protocol: DMDM+4.5g/L glucose+1% GluMax+10% FBS+2%P/S

extract_protocol:
Zymo RNA kit
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: 5pM miR-690 mimic/0.5x106 cells

tag: cell line

value: adipocyte

tag: passage


title: miR-690oe 3


title: miR-690oe 2


title: miR-690oe 1


title: Con 1

source_name: 3T3-L1 adipocytes


title: Con 3


title: Con 2


title: AR_38_Perigastric_LN

description: Chromatin IP for AR in Sample 38_Perigastric_LN

source_name: AR ChIP Sample 38 Perigastric lymph node


title: Con 4


title: H3K27ac_45_Hilar_LN

description: Chromatin IP for H3K27ac in Sample 45_Hilar_LN

source_name: H3K27ac ChIP Sample 45 Hilar lymph node


title: eGFP 2

city: Saint Louis

first: Jonathan

last: Kipnis

state: MO

address: 4515 McKinley Ave

department: Pathology and Immunology

institute: Washington University in Saint Louis

description:
RNA was isolated from LECs sorted from a pool of 10 mouse brain meninges
mouse_old_gfp_v_vegfc_norm_counts_sd2  (SL324045)

data_processing:
Raw FASTQ sequencing reads were chastity filtered to remove clusters having outlying intensity corresponding to bases other than the called base.
Filtered reads were assessed for quality using FastQC.
Filtered reads were trimmed using Trimmomatic to remove adapter sequences and low quality bases
Transcript-level abundances were calculated using Salmon.
Transcript-level abundances were summarized into gene-level counts with tximport.
Raw counts were then filtered to remove genes without at least  5 counts in at least 3 samples,  as well as genes with standard deviations of log transformed counts greater than 3 between replicates
The filtered raw counts were then normalized with DEseq2
Genome_build: mm10
Supplementary_files_format_and_content: processed data files are csv files containing gene x count matrices of the DEseq2 normalized counts.

source_name: Mouse brain meninges

extract_protocol:
RNA was extracted using Arcturus PicoPure kit (Thermo Fisher)
All RNA sample processing (including linear RNA amplification and cDNA library generation) and RNA-seq was performed by HudsonAlpha Genomic Services Laboratory

tag: celltype

value: meningeal lymphatic endothelial cells

value: 20-24 months

value: male

tag: treatment

value: 5 uL of AAV1-CMV-eGFP


title: mVEGF-C 2

description:
RNA was isolated from LECs sorted from a pool of 10 mouse brain meninges
mouse_old_gfp_v_vegfc_norm_counts_sd2  (SL324044)

tag: age

value: 5 uL AAV1-CMV-mVEGF-C


title: Tissue_and_splenic_Tregs_hashed

city: Boston

first: CBDM

last: Lab

state: MA

address: 77 Avenue Louis Pasteur

department: Microbiology and Immunobiology

institute: Harvard Medical School

data_processing:
CellRanger software (v3.1.0) (10X Genomics) (default parameters) using the mm10 genome.
Hashtag count matrices were obtained from CITE-Seq-Count package (https://zenodo.org/record/2590196) (default parameters).
Genome_build: mm10
Supplementary_files_format_and_content: 10X Genomics Filtered Gene BC Barcodes
Supplementary_files_format_and_content: 10X Genomics Filtered Gene BC Genes
Supplementary_files_format_and_content: 10X Genomics Filtered Gene BC Matrix
Supplementary_files_format_and_content: Metadata (including in particular the hashtag counts and HashID for each cell ID)

source_name: CD4 Treg

extract_protocol:
Tregs from VAT, liver, skin and spleen of a 20-week-old Pparg-Tdt.Foxp3-Gfp mouse were sorted and encapsulated using the Chromium Single Cell 5’ and V(D)J platform (10X Genomics).
RNA libraries were prepared for sequencing using standard 10X Genomics Single Cell 5′ Reagent Kit

value: B6 Pparg-Tdt.Foxp3-Gfp

tag: tissue

value: VAT, Liver, Skin and Spleen

value: CD4 Treg

value: Male

value: 20 weeks


title: Tissue_and_splenic_Tregs_hashed_TCR

data_processing:
CellRanger software (v3.1.0) (10X Genomics) (default parameters).
Genome_build: vdj_GRCm38_alts_ensembl-3.1.0
Supplementary_files_format_and_content: Consensus sequence annotations
Supplementary_files_format_and_content: Contig annotations

extract_protocol:
Tregs from VAT, liver, skin and spleen of a 20-week-old Pparg-Tdt.Foxp3-Gfp mouse were sorted and encapsulated using the Chromium Single Cell 5’ and V(D)J platform (10X Genomics).
RNA libraries were prepared for sequencing using standard 10X Genomics Single Cell V(D)J Reagent Kit

tag: strain


title: Spleen PPARGg low Tregs

data_processing:
CellRanger software (v2.1.0) (10X Genomics) (default parameters) using the mm10 genome.
Genome_build: mm10
Supplementary_files_format_and_content: 10X Genomics Filtered Gene BC Barcodes
Supplementary_files_format_and_content: 10X Genomics Filtered Gene BC Genes
Supplementary_files_format_and_content: 10X Genomics Filtered Gene BC Matrix

extract_protocol:
Tregs from spleens of two or three 8-week-old Pparg-Tdt.Foxp3-Gfp mice were pooled, sorted and encapsulated using the Chromium Single Cell 3’ v2 platform
RNA libraries were prepared for sequencing using standard 10X Genomics Single Cell 3′ Reagent Kit (V2 chemistry)

value: Spleen

tag: cell type

value: 8 weeks


title: mVEGF-C 1

description:
RNA was isolated from LECs sorted from a pool of 10 mouse brain meninges
mouse_old_gfp_v_vegfc_norm_counts_sd2 (SL324042)


title: eGFP 1

description:
RNA was isolated from LECs sorted from a pool of 10 mouse brain meninges
mouse_old_gfp_v_vegfc_norm_counts_sd2  (SL324043)


title: AR_40_lung

description: Chromatin IP for AR in Sample 40_lung

source_name: AR ChIP Sample 40 lung

value: metastatic castration resistant prostate cancer

value: lung


title: Spleen PPARg negative Tregs


title: NRVCs control_ssRNA_seq_rep1

city: Guangzhou

first: Youchen

last: Yan

state: Guangdong

address: No.58 Zhongshan Road II

institute: The First Affiliated Hospital, Sun Yat-sen University

data_processing:
For strand-specific RNA-seq , adaptor sequences were trimmed from raw RNA-seq data using cutadapt (v2.0.dev0).
Fragments were aligned to the rat genome (Rattus_norvegicus.Rnor_6.0) using STAR (v2.4.2a) with ENCODE standard options for long RNA-seq pipeline and the Ensembl transcriptome (Rattus_norvegicus.Rnor_6.0.94) as a reference.
We generated read counts using featureCounts (v1.6.4) with default options
Genome_build: Rattus_norvegicus.Rnor_6.0

source_name: Neonatal rat ventricular cardiomyocytes

growth_protocol: NRVCs were isolated by enzymatic disassociation repeatedly. All isolated cells were plated for 1 hours to remove non-cardiomyocytes. Non-adherent cells were then plated on 0.5% gelatin-coated plates and cultured in DMEM high glucose medium (Gibco) supplemented with sodium pyruvate(1X), GlutaMAX(1X) and 10% fetal bovine serum with antibiotics.

extract_protocol:
Cell total RNA was isolated using Trizol reagent.
Ribosomal RNA was depleted from the samples using NEBNext rRNA Depletion Kit (Human/Mouse/Rat) protocol (NEB, #E6310). Purified RNA was directly processed with the NEBNext ®  Ultra ™ II Directional RNA Library Prep Kit for Illumina (NEB, #7760)  and converted into libraries.
strand-specific RNA-seq

treatment_protocol: NRVCs treated with PE (20uM)) for 24h

value: Sprague-Dawley

value: cardiac ventricle

value: post natal day 2

value: untreated


title: NRVCs treated with PE_ssRNA_seq_rep2

value: treated with PE for 24 hours


title: NRVCs treated with PE_ssRNA_seq_rep1


title: NRVCs control_ssRNA_seq_rep2


title: NRVCs control_Ribo_seq_rep1

description: Ribosome protected RNA fragment

data_processing:
Library strategy: Ribo-seq
For Ribo-seq data, cutadapt (v2.0.dev0) was used to trim the relevant adapter sequence and remove the low-quality reads as well as filter reads the outside of 24-35 nt range.
Reads aligning to rRNA were removed using bowtie (v1.1.2) with default parameters. The remaining reads were then mapped to the rat genome (Rattus_norvegicus.Rnor_6.0) using STAR (v2.4.2a) with default options except for the following: --outFilterMismatchNmax 2, --outFilterMultimapNmax 5, --outFilterMatchNmin 16, --alignEndsType EndToEnd.
ORFs identification and quantification was performed according to the RiboCode (version 1.2.10) manual with the following parameters: -l no -m 10 -A CTG,GTG,TTG -g -b. The translation efficiency (TE) of ORFs were then calculated by Ribodiff (v0.2.2) analysis.
Genome_build: Rattus_norvegicus.Rnor_6.0

extract_protocol:
For ribosome profiling from cell, samples were snap frozen in liquid nitrogen after washed with ice-cold PBS containing 100 µg/ml cycloheximide. Then one 10-cm dish of cells were harvested in 400 µL ice-cold mammalian polysome buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2, with 1 mM DTT, 1% Triton X-100, 0.1% NP-40, 500U/mL RNase Inhibitor, 1× EDTA-free Complete protease inhibitors, 1U/µl DNase I and 100 µg/ml cycloheximide). After dispersal of the cells, the lysate was passed ten times through a 26 gauge needlet, cleared by centrifugation at 20,000g for 5 min. About 120 µl of the supernatant was digested with 3 µl of RNase I (Life Technologies, AM2294) for 45 min at room temperature. SUPERase•In RNase Inhibitor (Life Technologies, AM2694) was then added to stop the reaction. Resulting monosomes were purified with MicroSpin S-400 HR columns (GE  Healthcare, 27-5140-01). And the RNA was isolated from the flow-through (approximately 125 µl) using 1 mL Trizol. To enrich the RFPs (ribosome footprints) , the RNA samples was subjected to the Ribo-Zero Gold rRNA Removal Kit (Illumina, MRZG12324) to remove ribosomal RNA fragments and separated them on a 17% 7.5M Urea-PAGE gel. The size range from 26 nt to 30 nt, defined by loading with 14-30 ssRNA Ladder Marker (Takara, #3416), was cut out.
For Ribosome profiling, the purified RNA was end-repaired and then processed with the in house small RNA sequencing protocol and converted into libraries. Brifely, the RNA fragments was lingated to a pre-adenylated DNA linkers and followed a 5' RNA adaptor. After reverse transctiption, the cDNA was amplified and sequenced with Illumina X ten platform.

value: ventricular cardiomyocytes


title: NRVCs control_Ribo_seq_rep2

description: Ribosome protected RNA fragment


title: NRVCs treated with PE_Ribo_seq_rep1

description: Ribosome protected RNA fragment


title: NRVCs treated with PE_Ribo_seq_rep2

description: Ribosome protected RNA fragment


title: J01157-L1_S34_L007_R1_001

city: Kiel

first: Simonas

last: Juzenas

address: Am Botanischen Garten 11

department: Institute of Clinical Molecular Biology

country: Germany

institute: Christian-Albrechts-University of Kiel

data_processing:
docker run -v ~/excerpt/input:/exceRptInput \ -v ~/excerpt/results:/exceRptOutput \ -v ~/excerpt/hg38:/exceRpt_DB/hg38 \ -t rkitchen/excerpt \ ADAPTER_SEQ=AAAAAAAAAA \ MIN_READ_LENGTH=15 \ STAR_outFilterMatchNmin=15 \ RANDOM_BARCODE_LENGTH=4 \ RANDOM_BARCODE_LOCATION='-5p'
Genome_build: hg38
Supplementary_files_format_and_content: raw count and rpm

source_name: hemorrhoid tissue/anal canal

extract_protocol:
mirVana miRNA Isolation Kit (Ambion).
NEXTFLEX Combo-Seq Kit (Perkin Elmer).

tag: gender

value: male

tag: bmi

tag: condition

value: controls

value: hemorrhoid tissue/anal canal

tag: histological zone

value: anoderm region


title: WaGa [RNA]

city: Essen

first: Jan

last: Gravemeyer

address: Universitätstraße 1

institute: DKZF

description: mRNA expression was analyzed using the nCounter PanCancer Pathways Panel

hyb_protocol: CodeSet hybridization is achieved by the application of probe pairs that consist of a reporter probe, which carries the color code signal on it’s 5’ end, and a capture probe which carries biotin on it’s 3’end.

scan_protocol: not applicable

data_processing: Raw data was extracted from the nCounter RCC Collector file using the nSolver software and analyzed in R. Gene expression was normalized by using the four most stably expressed microRNAs identified by the GeNorm algorithm

source_name: cell line

label_protocol: mRNA sample preparation was done according to the manufacture’s instructions (NanoString)

growth_protocol: The vMCC (MCC13, MCC26 ) and cMCC (WaGa, PeTa, MKL-1, MKL-2) cell lines were maintained in RPMI-1640 (Pan Biotech, Aidenbach, Germany) supplemented with 10% fetal bovine serum (Sigma, St. Lous, MO, USA) and 1% penicillin/streptomycin (Pan Biotech, Aidenbach, Germany). The SCC cell lines (Met-1, HSC-1) were maintained in DMEM (Pan Biotech, Aidenbach, Germany), supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.

extract_protocol: RNA was isolated using the PeqGOLD total RNA kit (Peqlab, Erlangen, Germany).

tag: sample type

value: classical Merkel cell carcinoma


title: MKL-1 [RNA]


title: PeTa [RNA]


title: MCC26 [RNA]

value: variant Merkel cell carcinoma


title: J01160-L1_S10_L002_R1_001

value: female

value: transition zone


title: J01159-L1_S25_L005_R1_001

value: cases

value: intestinal mucosa region


title: MCC13 [RNA]


title: Met-1 [RNA]

value: Squamous cell carcinoma


title: HSC-1 [RNA]


title: J01156-L1_S20_L004_R1_001


title: J01152-L1_S38_L008_R1_001


title: J01154-L1_S9_L002_R1_001


title: J01150-L1_S30_L006_R1_001


title: J01155-L1_S5_L001_R1_001


title: J01151-L1_S19_L004_R1_001


title: J01148-L1_S14_L003_R1_001


title: J01149-L1_S24_L005_R1_001


title: MKL-2 [RNA]


title: J01147-L1_S8_L002_R1_001


title: J01145-L1_S3_L001_R1_001


title: J01146-L1_S33_L007_R1_001


title: J01158-L1_S15_L003_R1_001


title: J01144-L1_S18_L004_R1_001


title: J01143-L1_S13_L003_R1_001


title: J01141-L1_S22_L005_R1_001


title: J01142-L1_S23_L005_R1_001


title: J01139-L1_S21_L005_R1_001


title: J01153-L1_S4_L001_R1_001


title: J01136-L1_S12_L003_R1_001


title: J01140-L1_S2_L001_R1_001


title: J01133-L1_S37_L008_R1_001


title: J01132-L1_S16_L004_R1_001


title: J01131-L1_S1_L001_R1_001


title: J01125-L1_S35_L008_R1_001


title: J01129-L1_S28_L006_R1_001


title: J01124-L1_S31_L007_R1_001


title: J01128-L1_S27_L006_R1_001


title: J01126-L1_S32_L007_R1_001


title: J01123-L1_S26_L006_R1_001


title: RNA-seq_757509_BL

city: Houston

first: FENG

last: WANG

state: TX

address: 1881 East Road

institute: UT MD Anderson Cancer Center

data_processing:
Raw sequencing data from Illumina platform were converted to fastq files and aligned to the reference genome (hg19) using the Spliced Transcripts Alignment to a Reference (STAR) algorithm.
HTSeq-count was then utilized to generate the raw counts for each gene.
Raw counts were then analyzed by DESeq2 for data processing, normalization and differential expression analysis following standard procedures.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text file include normalized counts for each sample

source_name: Bone Marrow

extract_protocol: Strand specific RNA sequencing libraries were constructed using the Illumina TruSeq RNA Access Library Prep Kit (Illumina, San Diego, CA) according to the manufacturer’s protocol.

tag: patient diagnosis

value: AML

value: Bone Marrow

tag: idh1/2 genotype

value: IDH1/IDH2 mutant

tag: timepoint

value: BL

tag: treated with

value: IDH inhibitors


title: RNA-seq_974025_REL

value: REL


title: RNA-seq_4259035_BL


title: RNA-seq_4235533_BL


title: RNA-seq_4128869_BL


title: RNA-seq_2620771_BL


title: RNA-seq_2616045_BL


title: RNA-seq_2512641_BL


title: J01138-L1_S17_L004_R1_001

value: male


title: J01137-L1_S7_L002_R1_001

value: controls


title: J01134-L1_S6_L002_R1_001

value: hemorrhoid tissue/anal canal


title: RNA-seq_2502699_BL


title: RNA-seq_2498789_BL


title: J01135-L1_S29_L006_R1_001


title: RNA-seq_2463247_REL


title: J01130-L1_S36_L008_R1_001


title: RNA-seq_2480485_BL


title: J01127-L1_S11_L003_R1_001


title: RNA-seq_2430935_REL


title: RNA-seq_2430935_BL


title: RNA-seq_2450523_BL


title: RNA-seq_2410163_REL


title: RNA-seq_2410163_BL


title: RNA-seq_2382431_BL


title: RNA-seq_2366711_BL


title: RNA-seq_2370233_BL


title: RNA-seq_2361361_BL


title: RNA-seq_2297941_REL


title: RNA-seq_2297941_BL


title: RNA-seq_2297625_REL


title: RNA-seq_2583919_BL


title: RNA-seq_2297625_BL


title: RNA-seq_2296723_BL


title: RNA-seq_2295595_BL


title: RNA-seq_2283265_BL


title: RNA-seq_2280935_BL


title: RNA-seq_2264225_REL


title: RNA-seq_2292915_BL


title: RNA-seq_2463247_BL


title: RNA-seq_2292873_REL


title: RNA-seq_2419959_BL


title: RNA-seq_2263055_BL


title: RNA-seq_2260671_REL


title: RNA-seq_2219711_BL


title: RNA-seq_2260553_BL


title: RNA-seq_2247707_BL


title: RNA-seq_2163337_BL


title: RNA-seq_2361405_BL


title: RNA-seq_2151503_BL


title: RNA-seq_2150901_BL


title: RNA-seq_2010157_BL


title: RNA-seq_1825001_REL


title: RNA-seq_1825001_BL


title: RNA-seq_1794247_BL


title: RNA-seq_2264225_BL


title: A32_S346

city: New Brunswick

first: Shin-Heng

last: Chiou

state: NJ

address: 195 Little Albany St, Room 3001

department: Medicine

institute: Rutgers Cancer Institute of New Jersey

data_processing:
STAR aligner (2.6.1d) was used to map the reads with default parameters against human genome reference GRCh38 (v21) from the UCSC genome browser
Mapped reads were sorted and indexed with samtools (1.4)
Gene expression was first quantified by counting reads mapped to genes with htseq-count (HTSeq 0.5.4p5) using the following settings: --stranded=no --type=exon --idattr=gene_name --mode=intersection-nonempty
Single-cell T cell states were analyzed with Seurat (3.1.4) packages in R using raw read counts
Genome_build: GRCh38 (v21)
Supplementary_files_format_and_content: tab-delimited raw count txt file

source_name: Resected tumors of lung cancer

growth_protocol: Ex vivo samples immediately prepared from resected tumor

extract_protocol:
T cells were isolated from tumor single cell suspensions by antibody staining followed by cell sorting on a 5-laser FACSAria Fusion (Stanford FACS Facility) purchased using funds from the Parker Institute for Cancer Immunotherapy. Tumor cell suspensions were stained in PBS with Zombie Aqua dye (Biolegend) for viability assessment. This was followed by staining in PBS with 2% FBS in Fc Blocking solution (Biolegend) plus the following antibodies: anti-CD4 (OKT4, Biolegend), anti-CD8 (SK1, Biolegend), anti-CD3 (OKT3, Biolegend), anti-CD45 (H130, Biolegend), anti-CD25 (BC96, Biolegend), anti-PD-1(EH12.2H7, Biolegend), anti-CD137 (4B4-1, BD Biosciences), anti-HLA-DR (L243, Biolegend). CD3+CD45+AquaZombie- cells were index sorted directly into 96-well plates preloaded with 4 uL of capture buffer, snap frozen on dry ice, and stored at -80C.
Full transcriptomes from FACS sorted T cells at the single-cell level were generated according to the previously reported procedures with some modifications (Picelli et al., 2014). First strand cDNA was then generated with Takara’s SMARTScribe Reverse Transcriptase kit according to manufacturer’s protocol (Takara Bio). Notable changes from the previously reported Smart-Seq2 RT step includes: 2 mM of dNTP and 2 M of oligo dT were included in the capture buffer; 1M of Betaine and additional 6 mM MgCl2 were included in the RT reaction buffer. The cDNA samples were then amplified with the KAPA Library Quantification kit for 22 – 25 cycles (Roche). We used 1 (of total 25/well) L of amplified cDNA for single-cell TCR-sequencing and thus bypassing the RT step as reported previously (Han et al., 2014). To proceed with scRNA-Seq, full-length cDNA samples were first cleaned up with 0.6 – 0.8x volume of precalibrated AMPure XP beads (Beckman Coulter) to exclude DNA fragments smaller than 500 base pairs. We used the automatic liquid handler Biomek FXP Automated Workstation (Beckman Coulter) in order to eliminate cell-to-cell variabilities. The quality of purified full-length cDNA was validated with the AATI Fragment Analyzer (Agilent). Subsequently, we used the measurements from the Fragment Analyzer in order to normalize the cDNA input with a Mantis liquid handler (Formulatrix). We then consolidated the cDNA samples into a 384-well plate (LVSD) with a Mosquito X1 liquid handler (TTP labtech). After transfer, Illumina sequencing libraries were prepared using a Mosquito HTS liquid handler (TTP labtech). We used only 0.4 uL (of total 23 uL) of cDNA per well to make the full transcriptome libraries with the Nextera XT DNA Library Preparation Kit (Illumina, FC-131-1096). We used custom-made i5 and i7 unique 8-bp indexing primers (IDT) to multiplex 384 wells in a single sequencing run. The libraries were amplified on a C1000 Touch™ Thermal Cycler with 384-Well Reaction Module (Bio-rad). We checked the pooled libraries with the Agilent 2100 Bioanalyzer (Stanford PAN facility) and acquired paired-end sequences (150bp x 2) on a Hiseq 4000 Sequencing System (Illumina) purchased with funds from NIH (S10OD018220) for the Stanford Functional Genomics Facility (SFGF).
Smart-Seq2 Method (Picelli et al., 2014)

treatment_protocol: Tumor dissociation followed immediately by FACS sort onto 96-well plates and -80C storage

value: CD45+CD3+ T cells

value: Resected tumors


title: A32_S315


title: A32_S354


title: A32_S361


title: A32_S338


title: A32_S337


title: A32_S328


title: RNA-seq_2210551_BL


title: A32_S313


title: A32_S353


title: A32_S344


title: RNA-seq_2152641_BL


title: A32_S332


title: A32_S312


title: A32_S351


title: A32_S327


title: A32_S350


title: A32_S358


title: A32_S311


title: A32_S343


title: A32_S331


title: A32_S326


title: A32_S345


title: A32_S342


title: A32_S348


title: A32_S349


title: A32_S310


title: A32_S335


title: A32_S341


title: A32_S325


title: A32_S318


title: A32_S334


title: A32_S330


title: A32_S317


title: A32_S323


title: A32_S316


title: A32_S333


title: A32_S319


title: A32_S293


title: A32_S283


title: A32_S254


title: A32_S273


title: A32_S309


title: A32_S253


title: A32_S282


title: A32_S263


title: A32_S264


title: A32_S299


title: A32_S281


title: A32_S271


title: A32_S262


title: A32_S324


title: A32_S280


title: A32_S270


title: A32_S250


title: A32_S291


title: A32_S239


title: A32_S298


title: A32_S307


title: A32_S294


title: A32_S289


title: A32_S268


title: A32_S279


title: A32_S260


title: A32_S292


title: A32_S261


title: A32_S290


title: A32_S238


title: A32_S287


title: A32_S237


title: A32_S267


title: A32_S305


title: A32_S251


title: A32_S278


title: A32_S258


title: A32_S297


title: A32_S236


title: A32_S247


title: A32_S248


title: A32_S296


title: A32_S276


title: A32_S303


title: A32_S246


title: A32_S266


title: A32_S275


title: A32_S295


title: A32_S284


title: A32_S274


title: A32_S245


title: A32_S235


title: A32_S302


title: A32_S223


title: A32_S255


title: A32_S257


title: A32_S211


title: A32_S189


title: A32_S199


title: A32_S165


title: A32_S153


title: A32_S210


title: A32_S222


title: A32_S188


title: A32_S198


title: A32_S177


title: A32_S164


title: A32_S187


title: A32_S175


title: A32_S265


title: A32_S221


title: A32_S163


title: A32_S151


title: A32_S186


title: A32_S220


title: A32_S150


title: A32_S232


title: A32_S207


title: A32_S161


title: A32_S185


title: A32_S206


title: A32_S172


title: A32_S148


title: A32_S217


title: A32_S205


title: A32_S229


title: A32_S171


title: A32_S183


title: A32_S147


title: A32_S159


title: A32_S216


title: A32_S228


title: A32_S204


title: A32_S194


title: A32_S170


title: A32_S146


title: A32_S182


title: A32_S158


title: A32_S231


title: A32_S215


title: A32_S227


title: A32_S203


title: A32_S193


title: A32_S157


title: A32_S181


title: A32_S202


title: A32_S156


title: A32_S226


title: A32_S191


title: A32_S155


title: A32_S213


title: A32_S180


title: A32_S190


title: A32_S178


title: A32_S154


title: A32_S131


title: A32_S121


title: A32_S90


title: A32_S70


title: A32_S141


title: A32_S102


title: A32_S130


title: A32_S101


title: A32_S89


title: A32_S129


title: A32_S119


title: A32_S140


title: A32_S100


title: A32_S69


title: A32_S110


title: A32_S139


title: A32_S88


title: A32_S68


title: A32_S145


title: A32_S78


title: A32_S118


title: A32_S87


title: A32_S77


title: A32_S138


title: A32_S109


title: A32_S117


title: A32_S67


title: A32_S127


title: A32_S108


title: A32_S97


title: A32_S86


title: A32_S76


title: A32_S116


title: A32_S85


title: A32_S136


title: A32_S126


title: A32_S66


title: A32_S106


title: A32_S84


title: A32_S135


title: A32_S65


title: A32_S94


title: A32_S83


title: A32_S125


title: A32_S114


title: A32_S93


title: A32_S133


title: A32_S73


title: A32_S82


title: A32_S72


title: A32_S104


title: A32_S113


title: A32_S123


title: A32_S81


title: A32_S122


title: A32_S132


title: A32_S92


title: A32_S103


title: A32_S91


title: A32_S115


title: A32_S41


title: A32_S71


title: A32_S24


title: A32_S40


title: A32_S7


title: A32_S55


title: A32_S39


title: A32_S134


title: A32_S124


title: A32_S30


title: A32_S23


title: A32_S61


title: A32_S54


title: A32_S22


title: A32_S53


title: A32_S29


title: A32_S46


title: A32_S38


title: A32_S21


title: A32_S20


title: A32_S45


title: A32_S13


title: A32_S4


title: A32_S51


title: A32_S3


title: A32_S18


title: A32_S12


title: A32_S60


title: A32_S2


title: A32_S11


title: A32_S50


title: A32_S17


title: A32_S14


title: A32_S10


title: A32_S49


title: A32_S9


title: A32_S43


title: A32_S1


title: A32_S33


title: A32_S57


title: A32_S25


title: A32_S19


title: A31_S302


title: A31_S347


title: A31_S283


title: A32_S37


title: A31_S282


title: A31_S335


title: A31_S345


title: A31_S313


title: A31_S324


title: A32_S26


title: A31_S301


title: A31_S323


title: A31_S355


title: A31_S354


title: A31_S333


title: A31_S279


title: A31_S311


title: A31_S343


title: A31_S310


title: A31_S298


title: A31_S357


title: A31_S342


title: A31_S289


title: A31_S309


title: A31_S314


title: A31_S297


title: A31_S276


title: A31_S341


title: A31_S329


title: A31_S318


title: A31_S350


title: A31_S312


title: A31_S291


title: A31_S328


title: A31_S274


title: A31_S306


title: A31_S286


title: A31_S339


title: A31_S280


title: A31_S327


title: A31_S295


title: A31_S331


title: A31_S262


title: A31_S212


title: A31_S228


title: A31_S219


title: A31_S271


title: A31_S261


title: A31_S227


title: A31_S251


title: A31_S275


title: A31_S270


title: A31_S211


title: A31_S250


title: A31_S260


title: A31_S238


title: A31_S305


title: A31_S226


title: A31_S259


title: A31_S237


title: A31_S268


title: A31_S249


title: A31_S269


title: A31_S225


title: A31_S338


title: A31_S267


title: A31_S284


title: A31_S247


title: A31_S217


title: A31_S235


title: A31_S216


title: A31_S234


title: A31_S209


title: A31_S200


title: A31_S215


title: A31_S266


title: A31_S244


title: A31_S233


title: A31_S232


title: A31_S248


title: A31_S222


title: A31_S265


title: A31_S201


title: A31_S221


title: A31_S231


title: A31_S243


title: A31_S197


title: A31_S242


title: A31_S220


title: A31_S254


title: A31_S230


title: A31_S263


title: A31_S253


title: A31_S205


title: A31_S241


title: A31_S223


title: A31_S229


title: A31_S199


title: A31_S213


title: A31_S204


title: A31_S169


title: A31_S186


title: A31_S165


title: A31_S177


title: A31_S196


title: A31_S185


title: A31_S168


title: A31_S150


title: A31_S195


title: A31_S184


title: A31_S214


title: A31_S183


title: A31_S194


title: A31_S154


title: A31_S193


title: A31_S159


title: A31_S175


title: A31_S146


title: A31_S191


title: A31_S152


title: A31_S162


title: A31_S180


title: A31_S167


title: A31_S171


title: A31_S179


title: A31_S172


title: A31_S155


title: A31_S151


title: A31_S161


title: A31_S170


title: A31_S83


title: A31_S187


title: A31_S105


title: A31_S166


title: A31_S148


title: A31_S127


title: A31_S103


title: A31_S136


title: A31_S158


title: A31_S126


title: A31_S153


title: A31_S135


title: A31_S101


title: A31_S117


title: A31_S100


title: A31_S145


title: A31_S89


title: A31_S142


title: A31_S78


title: A31_S134


title: A31_S115


title: A31_S122


title: A31_S110


title: A31_S87


title: A31_S98


title: A31_S76


title: A31_S132


title: A31_S121


title: A31_S140


title: A31_S131


title: A31_S125


title: A31_S85


title: A31_S108


title: A31_S74


title: A31_S84


title: A31_S139


title: A31_S40


title: A31_S106


title: A31_S56


title: A31_S118


title: A31_S29


title: A31_S130


title: A31_S71


title: A31_S18


title: A31_S55


title: A31_S28


title: A31_S9


title: A31_S17


title: A31_S62


title: A31_S38


title: A31_S70


title: A31_S46


title: A31_S75


title: A31_S37


title: A31_S26


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source_name: FACS-sorted peripheral blood T cells

growth_protocol: Ex vivo samples immediately prepared from FACS sort

extract_protocol:
Recombinant HLA-A*02 monomer with UV exchangeable peptide were either synthesized as previously described (Altman and Davis, 2003) or purchased commercially (Biolegend). UV peptide exchange was performed over 20 minutes with 1 mM of peptide in PBS using Strategene UV Stratalinker 2400. Streptavidin conjugated fluorophore was added incrementally the following day for a final 4:1 molar ratio of MHC:streptavidin. Tetramer staining was performed in PBS plus 2% FBS in Fc Blocking solution (Biolegend) at room temperature for 1 hour. For peripheral blood samples, cells were subsequently stained with anti-TCR (B1, Biolegend), anti-CD19 (H1B19, Biolegend), anti-CD14 (M5E2, Biolegend), anti-CD3 (OKT3, Biolegend), anti-CD4 (RPA-T4, Biolegend), anti-CD8 (HIT8a, Biolegend), and live/dead near-IR dye (Invitrogen). Tet+CD8+CD3+CD45+AquaZombie- cells were index sorted directly into 96-well plates preloaded with 4 uL of capture buffer, snap frozen on dry ice, and stored at -80C.
Full transcriptomes from FACS sorted T cells at the single-cell level were generated according to the previously reported procedures with some modifications (Picelli et al., 2014). First strand cDNA was then generated with Takara’s SMARTScribe Reverse Transcriptase kit according to manufacturer’s protocol (Takara Bio). Notable changes from the previously reported Smart-Seq2 RT step includes: 2 mM of dNTP and 2 M of oligo dT were included in the capture buffer; 1M of Betaine and additional 6 mM MgCl2 were included in the RT reaction buffer. The cDNA samples were then amplified with the KAPA Library Quantification kit for 22 – 25 cycles (Roche). We used 1 (of total 25/well) L of amplified cDNA for single-cell TCR-sequencing and thus bypassing the RT step as reported previously (Han et al., 2014). To proceed with scRNA-Seq, full-length cDNA samples were first cleaned up with 0.6 – 0.8x volume of precalibrated AMPure XP beads (Beckman Coulter) to exclude DNA fragments smaller than 500 base pairs. We used the automatic liquid handler Biomek FXP Automated Workstation (Beckman Coulter) in order to eliminate cell-to-cell variabilities. The quality of purified full-length cDNA was validated with the AATI Fragment Analyzer (Agilent). Subsequently, we used the measurements from the Fragment Analyzer in order to normalize the cDNA input with a Mantis liquid handler (Formulatrix). We then consolidated the cDNA samples into a 384-well plate (LVSD) with a Mosquito X1 liquid handler (TTP labtech). After transfer, Illumina sequencing libraries were prepared using a Mosquito HTS liquid handler (TTP labtech). We used only 0.4 uL (of total 23 uL) of cDNA per well to make the full transcriptome libraries with the Nextera XT DNA Library Preparation Kit (Illumina, FC-131-1096). We used custom-made i5 and i7 unique 8-bp indexing primers (IDT) to multiplex 384 wells in a single sequencing run. The libraries were amplified on a C1000 Touch™ Thermal Cycler with 384-Well Reaction Module (Bio-rad). We checked the pooled libraries with the Agilent 2100 Bioanalyzer (Stanford PAN facility) and acquired paired-end sequences (150bp x 2) on a Hiseq 4000 Sequencing System (Illumina) purchased with funds from NIH (S10OD018220) for the Stanford Functional Genomics Facility (SFGF).
Smart-Seq2 Method (Picelli et al., 2014)

treatment_protocol: PBL enrichment followed immediately by FACS sort onto 96-well plates and -80C storage

value: CD45+CD3+CD8+Tet+ T cells

value: Peripheral blood


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title: Vira_A11vr_S198


title: path_He65_S153


title: Vira_A11vr_S214


title: Vira_A11vr_S219


title: Vira_A11vr_S197


title: Vira_A11vr_S227


title: Vira_A11vr_S207


title: Vira_A11vr_S203


title: Vira_A11vr_S202


title: Vira_A11vr_S230


title: Vira_A11vr_S216


title: Vira_55vr_S104


title: Vira_55vr_S117


title: Vira_A11vr_S190


title: Vira_A11vr_S189


title: Vira_A11vr_S223


title: Vira_55vr_S116


title: Vira_A11vr_S201


title: Vira_55vr_S58


title: Vira_55vr_S70


title: Vira_55vr_S139


title: Vira_55vr_S138


title: Vira_A11vr_S213


title: Vira_55vr_S115


title: Vira_A11vr_S200


title: Vira_55vr_S81


title: Vira_55vr_S68


title: Vira_55vr_S93


title: Vira_55vr_S92


title: Vira_55vr_S69


title: Vira_55vr_S56


title: Vira_55vr_S114


title: Vira_55vr_S125


title: Vira_55vr_S78


title: Vira_55vr_S124


title: Vira_55vr_S136


title: Vira_55vr_S67


title: Vira_55vr_S90