title: SCR AMO 36 h - H3K9me3 rep2

city: Rochester

first: jinlong

last: lu

address: 213 Hutchinson Hall

institute: University of Rochester

data_processing:
Trim with paird-mode using trim_galore
Mapping with paird-mode using bowtie2
convert sam file to bam file using samtools
remove PCR duplicates using picard-tools
generate bw file from bam file using bamCoverage
Genome_build: mm9
Supplementary_files_format_and_content: bigwig

source_name: cj9 mESCs_SCR AMO 36 h - H3K9me3

growth_protocol: Wild-type (cj9) and infected embryonic stem cells (ESCs) were cultured on gelatin coated plates in standard ESC medium consisting of DMEM (Cellgro) supplemented with 15% heat-inactivated fetal bovine serum (Hyclone), 1% Glutamax (GIBCO), 1% Penicillin/Streptomycin (Cellgro), 1% nucleoside (Millipore), 0.1mM  2-mercaptoethanol  (GIBCO),  1%  MEM  nonessential  amino  acids  (Cellgro),  and  1000U/ml  recombinant leukemia inhibitory factor (Millipore).

extract_protocol:
Treated cells were harvested by trypsinization and rinsed twice by PBS. And cells were crosslinked by 1% FMA in 1 ml solution with rotation at RT for 10 min. And crosslinking was stopped by addition of 125 mM glycine. Cells were washed with PBS twice and then treated with 200 μl lysis buffer (50 mM Tris-HCl pH 8.1, 10 mM EDTA, 1% SDS, supplied with 1 mM DTT and 1 mM PMSF) on ice for 10 min. Sonication was performed with 10 s on, 20 s off for 5 cycles with 25% amplification. Cells were spun down at 14,000 rpm at 4℃ for 10 min, and supernatant was collected for immunoprecipitation. For ChIP, 1 μl H3K9me3 antibody was added into the sample and incubated at 4℃ overnight, after washing with Low Salt IP Wash Buffer (25 mM Tris-HCl pH 8.1, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate), High Salt IP Wash Buffer (25 mM Tris-HCl pH 8.1, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate), LiCl IP Wash Buffer (10 mM Tris-HCl pH 8.1, 250 mM LiCl, 1 mM EDTA, 0.5% NP-40, 0.1% Na-deoxycholate) and TE buffer (10 mM Tris-HCl pH 8.1, 1 mM EDTA), ChIPed DNA were eluated twice by IP Elution solution (50 mM Tris-HCl pH 8.1, 1% SDS, 1 mM EDTA and supplied with 100 mM NaHCO3, 250 mM NaCl, 80 μg/ml Proteinase K) at 65℃ for 20 min. After reverse crosslinking at 65℃ for 6-16 h, DNA were extracted by AMPure XP beads (Beckman Coulter).
ChIP-seq were prepared by library preparation modules (New England Biolabs)

treatment_protocol: The cells were treated with L1 or SCR control AMO probes using Mouse Embryonic Stem Cell NucleofectorTM Kit (VPH-1001) via Nucleofector 2b Device (Amaxa Nucleofector II Electroporation Machine (Lonza)).

tag: strain background

tag: cell line

value: embryonic stem cells (ESCs)

value: SCR AMO 36 h

tag: chip antibody


title: 5_92a_2 SCR 3d.rma-gene-full-Signal

status: Public on Dec 14 2020

type: RNA

city: Malmö

first: Charlotte

last: Ling

address: Jan Waldenströms gata 35, CRC 91:12

department: Clinical Sciences

country: Sweden

institute: Lund University

hyb_protocol: according to the manufacturer’s recommendations

data_processing: Expression Console Software v1.4.1.46 was used for quality analyses and data were processed using Robust Multi-array Analysis (RMA) and log2 transformed. Paired t-tests were used when comparing Control vs siVPS39.

label: biotin

molecule: total RNA

organism: Homo sapiens

source_name: muscle cells

growth_protocol: Briefly, myoblasts were cultured in growth medium (HAM’s/F10 supplemented with 20% fetal bovine serum (FBS), 1% penicillin/streptomycin and 1% Amphotericin B). To induce differentiation, cells were incubated in differentiation medium 1 (DMEM with 1.0 g/l glucose supplemented with 10% FBS and 1% penicillin/streptomycin) for 2 days, followed by differentiation medium 2 (DMEM with 4.5 g/l glucose supplemented with 2% horse serum and 1% penicillin/streptomycin) until cells were fully differentiated, as determined by visual confirmation of myotube formation (>3 nuclei per myotube in approximately 70% of cells) on day 7.

extract_protocol: RNA from VPS39-silenced cells was extracted using RNeasy mini kit (Qiagen #74104)

treatment_protocol: Primary human myoblasts (at 70-90% confluence) were transfected with ON-TARGETplus human set of four siRNA molecules (Dharmacon, Lafayette, CO, USA) targeting VPS39 (LQ-014052-01) or a negative control (Non-targeting plus #D-001810-10).

tag: genotype/variation

value: Control


title: HH-041

city: Oxford

first: Zhiyuan

last: Hu

address: John Radcliffe Hospital/Headley Way

department: MRC Weatherall Institute of Molecular Medicine

country: United Kingdom

institute: University of Oxford

hyb_protocol: 70 μL hybridisation buffer was added to Reporter CodeSet to make the master mix. Master Mix, total RNA and water were added to PCR tubes to be hybridised at 65°C for 19 hours.

scan_protocol: Not provided

data_processing: Processed by using nSolver.

source_name: Ovarian tumour

label_protocol: Not provided

extract_protocol: Frozen tumor piece was placed into a tube containing 500 μl RLT buffer from RNeasy kit (QIAGEN) and one Retsch 6 mm steel core bead. Tubes were placed into well adapters of a Tissuelyser II (QIAGEN) and tissues were lysed at 15 Hz for 2 min. Tubes were centrifuged briefly and 320 μl was removed for subsequent RNA extraction using the RNeasy kit (QIAGEN) according to the manufacturer’s instructions. RNA concentrations were quantified using the Bioanalyzer system (Agilent).

tag: pfs

tag: histology

value: Serous

tag: age

tag: grade

value: high

tag: Stage

value: late


title: 320425: EtOH_12

type: SRA

city: Charlottesville

first: Matthew

last: Devall

state: Virginia

address: 1335 Lee Street

country: USA

institute: University of Virginia

library_source: transcriptomic

data_processing:
Base calls were generated in real-time on the NovaSeq6000 instrument (RTA 3.1.5); (2) demultiplexed, unaligned BAM files produced by Picard ExtractIlluminaBarcodes and IlluminaBasecallsToSam are converted to FASTQ format using SamTools bam2fq (v1.4); (3) sequence read and base quality are checked using the FASTX-toolkit (v0.0.13); (4) sequences are aligned to GRCh38 with reference transcriptome GENCODE release 29 using STAR (v2.6.1d
Gene level expression quantification is generated with RNA-SeQC (v2.3.3) and RSEM (v1.3.1). RPKM and TPM for transcript isoforms are quantitated on the merged lane-level data with RSEM (v1.3.1). Gene-level RPKMs are quantitated with RNA-SeQC (v2.3.3) on a standard collapsed reference annotation
Genome_build: GrCh38, GENCODE release 29
Supplementary_files_format_and_content: "Pheno.csv" contains sample metadata, "Etoh_Counts.csv" contains raw counts, "SummaryStats.csv" contains the number and percentages of reads mapped for each samples

molecule: polyA RNA

source_name: Colon Organoid

growth_protocol: Normal 3D colon organoids included in the study were developed from biopsies of either distal or proximal colon using a modification of the method described by Sato et al. After isolation, colon crypts were embedded in Matrigel, and cell lines were established in growth media that included: advanced DMEM/F-12, 100U/ml penicillin, 100μg/ml streptomycin, 10mM Hepes, 1x N2, 1x B27, 2mM GlutaMAX, 1mM N-acetyl-cysteine, 10mM gastrin, 50% L-WRN conditioned media, 500nM A83-01, 10uM SB202190, 10mM nicotinamide, 50ng/ml EGF, 10μM Y27632. Colon organoids were grown and passaged as needed in 48-well culture plates.

extract_protocol:
For RNA isolation, media was gently removed and 200μl RNA lysis Solution RA1 with TCEP (Machery-Nagel; Cat No: 740902.50) was added to each well. The lysate was collected in a 1.5ml Eppendorf tube. Each tube was vortexed vigorously and placed on ice. Further steps were carried out according to manufacturer’s protocol of the Machel-Nagel RNA XS kit. RNA was checked for quality using the Agilent 4200 Tapestation. Samples with RIN > =8.5 were considered for library preparation and sequencing using the Illumina NovoSeq
Total RNA is normalized to 7.5ng/ul in a total volume of 50ul on the Perkin Elmer Janus Workstation (Perkin Elmer, Janus II).  Poly-A selection and cDNA synthesis are performed using the TruSeq Stranded mRNA kit as outlined by the manufacturer (Illumina, cat#RS-122-2103).  All steps are automated on the Perkin Elmer Sciclone NGSx Workstation to reduce batch to batch variability and to increase sample throughput.  Final RNASeq libraries are quantified using the Quant-it dsDNA High Sensitivity assay, and library insert size distribution is checked using a fragment analyzer (Advanced Analytical; kit ID DNF474).  Samples where adapter dimers constitute more than 4% of the electopherogram area are failed prior to sequencing.  Technical controls (K562,Thermo Fisher Scientific, cat# AM7832), are compared to expected results to ensure that batch to batch variability is minimized.  Successful libraries are normalized to 10nM for submission to sequencing.
Libraries were multiplexed and sequenced on Illumina Novaseq6000 to generate 100bp PE reads.

treatment_protocol: For this experiment, cells were treated with fresh growth media plus ethanol (Sigma Aldrich; Cat No: E7023) (2μl per 1ml of growth media), or fresh growth media plus 2μl cell culture grade distilled water as a control. Media (with or without ethanol) was replaced every 24 hours for 3 days in order to replace evaporating ethanol.

tag: cell type

value: Colon Organoid

tag: gender

value: Female

tag: treatment

value: EtOH

tag: tissue

value: Distal

tag: sample type

value: RNA-sequencing


title: 320420: CTL_14

value: CTL


title: SCR AMO 36 h - H3K9me3 rep1

state: NY

library_source: genomic

molecule: genomic DNA

organism: Mus musculus


title: HH-040


title: erk7 fed rep4

city: Helsinki

first: Krista

last: Kokki

address: Viikinkaari 9, Biocenter 1, Institute of Biotechnology P.O.Box. 56

department: Department of Biosciences (Genetics)

country: Finland

institute: University of Helsinki

description:
all_comparisons.txt
S 8

data_processing:
Sequenced reads were trimmed with Trimmomatic with parameters ILLUMINACLIP:TruSeq3-SE:2:30:10 LEADING:20 TRAILING:20 SLIDINGWINDOW:4:15 MINLEN:36
Sequences were mapped using TopHat (v2.1.0) to dmel R6.10 with parameter -p 4 and quantified with HTSeq (v2.7.6) with parameters -m HTSeq.scripts.count -m union -s reverse -a 10 -t exon -i Parent
Differentially expressed genes were calculated with R package limma with Benjamini-Hochberg correction, with genes with low counts (cpm<1 in more than one replicate) filtered
Genome_build: Flybase Drosophila melanogaster R6.10
Supplementary_files_format_and_content: tab delimited.txt file includes read counts for expressed genes for all samples and their statistics

organism: Drosophila melanogaster

source_name: whole larvae

growth_protocol: Control and ERK7 mutant flies were kept in egg laying champers with apple juice plates and yeast paste for 3 hours. 24 hours later the 1st instar larvae were transferred to standard lab food. 48 hours later larvae were transferred to either PBS + 0.5% agarose food or a fresh standard lab food. 6 hours later, larvae were collected from both food types for RNA extraction.

extract_protocol:
Larvae were frozen in liquid nitrogen and homogenized with a pestle and mortar in lysis buffer and vortexed. Samples were frozen for complete lysis of the tissue. Total RNA was extracted with Nucleospin RNA II kit according to manufacturer's protocol. Elution was done with water and the samples stored at -80°C. Library construction was performed by DNA sequencing and genomics lab at the Institute of Biotechnology with the following protocol: the total RNA samples were first treated with Dnase I. Then the mRNA was enriched by using the oligo(dT) magnetic beads. Mixed with the fragmentation buffer, the mRNA was fragmented into short fragments. The first stand of cDNA was synthesized by using random hexamer-primer. Buffer, dNTPs, RNase H and DNA polymerase I were added to synthesize the second strand. The double strand cDNA was purified with magnetic beads. End reparation and 3'-end single nucleotide A addition was performed. Finally, sequencing adaptors were ligated to the fragments and fragment enrichment was done by PCR amplification.
Library construction was performed by DNA sequencing and genomics lab at the Institute of Biotechnology with the following protocol: mRNA was enriched by using the oligo(dT) magnetic beads. Mixed with the fragmentation buffer, the mRNA was fragmented into short fragments. The first stand of cDNA was synthesized by using random hexamer-primer. Buffer, dNTPs, RNase H and DNA polymerase I were added to synthesize the second strand. The double strand cDNA was purified with magnetic beads. End reparation and 3'-end single nucleotide A addition was performed. Finally, sequencing adaptors were ligated to the fragments and fragment enrichment was done by PCR amplification.

tag: genotype

value: erk7 mutant

value: whole larvae

tag: developmental stage

value: 3rd instar larvae


title: HH-042


title: 320419: EtOH_15

value: Male


title: HH-039

value: low


title: 1_92a SCR 3d.rma-gene-full-Signal

scan_protocol: according to the manufacturer’s recommendations


title: erk7 fed rep2

description:
all_comparisons.txt
S 6


title: L1 AMO 36 h - H3K9me3 rep2

source_name: cj9 mESCs_L1 AMO 36 h - H3K9me3

value: embryonic stem cells (ESCs)

value: L1 AMO 36 h


title: 320422: CTL_13


title: 320418: CTL_15


title: HH-038

value: early


title: L1 AMO 36 h - H3K9me3 rep1


title: erk7 fed rep1

description:
all_comparisons.txt
S 5


title: 320417: EtOH_16


title: erk7 fed rep3

description:
all_comparisons.txt
S 7


title: adult_rep2: ex-vivo-microglia-naive-A7

city: Cambridge

first: Alex

last: Delbridge

state: MASSACHUSETTS

address: 225 binney street

institute: Biogen

data_processing:
Steps below for 10X Genomics single cell RNA-seq experiments TST11200, TST11247
"mkfastq" command from cellranger v1.3.0 was used to generate fastq from bcl files, with default parameters
"count" command from cellranger v1.3.0 was used to generate UMI table from fastq, with default parameters
custom R code was used to transfer sparse matrix format of "filtered_gene_bc_matrices" from the "count" output into full-matrix UMI table in csv format.
Genome_build: mm10
Supplementary_files_format_and_content: fastq: sequencing results. csv: UMI count table per barcode per gene, in full-matrix format.

source_name: microglia

extract_protocol:
Microglia were isolated under ice cold conditions. Pure populations of microglia were obtained by cell sorting for live CD11b positive, CD45 intermediate cells. Approximately equal numbers of live cells (~1000 cells per sample) were used as input for 10x chromium sequencing
10X Genomics 3' Gene Expression Kit v2

value: microglia

tag: tissue origin

value: brain tissue, adult

tag: strain

tag: immunophenotype

value: CD11b high; CD45 intermediate

tag: study id

tag: rna-seq protocol

value: 10X Genomics 3' Gene Expression Kit v2


title: control fed rep4

description:
all_comparisons.txt
S 4

value: wild type


title: HH-037

tag: pfs

value: Serous

value: high

value: late


title: 320416: CTL_16

value: Colon Organoid

value: CTL

value: Distal

value: RNA-sequencing


title: HH-036

tag: histology


title: 320415: EtOH_17

value: Female

value: EtOH

tag: age


title: control fed rep2

description:
all_comparisons.txt
S 2

value: whole larvae

value: 3rd instar larvae


title: slice_1wk_rep2: brain-slice-microglia-1-wk-C7

source_name: brain slice microglia

value: brain slice microglia

value: cultured brain slice after 1 week


title: slice_1wk_rep1: brain-slice-microglia-1-wk-B7


title: 320414: CTL_17


title: control fed rep1

description:
all_comparisons.txt
S 1


title: slice_3wks_rep2: brain-slice-microglia-3-wks-H1

value: cultured brain slice after 3 weeks


title: slice_1d_rep2: brain-slice-microglia-1d-F7

value: cultured brain slice after 1 day

value: CD11b high; CD45 intermediate


title: PSc

data_processing:
Sequencing reads were trimmed with Trim Galore! v0.3.1 (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) to remove adapters and low-quality bases.
Trimmed reads were mapped to a genome combining spike-in sequences and the mm9 mouse genome using BWA mem v.0.7.12.
PCR duplicates were removed using MarkDuplicate function of Picard v2.3.0 (http://broadinstitute.github.io/picard/). The overlapping regions were clipped by BamUtil v1.0.14 ClipOverlap.
Modified bases (CpGs only) were called by asTair 3.3.1 (https://pypi.org/project/asTair/3.3.1/) from reads with MAPQ over 10.
Regions known to be prone to mapping artifacts (https://sites.google.com/site/anshulkundaje/projects/blacklists) and known single nucleotide variants (http://epigenetics.hugef-research.org/data.php) of the E14 cell line were used to exclude those overlapping sites from subsequent analysis.
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text files include chr (first column), start position (second), end position (third), modification level (fourth), unmodified reads (fiveth) and modified reads (sixth)

source_name: E14 mESC

growth_protocol: E14 mouse embryonic stem cells (mESCs) were cultured on gelatin-coated plates in Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen) supplemented with 15% FBS (GIBCO), 2 mM L-glutamine (Gibco), 1% non-essential amino acids (Gibco), 1% penicillin/streptavidin (Gibco), 0.1 mM β-mercaptoethanol (Sigma), 1000 units/mL LIF (Millipore), 1 µM PD0325901 (Stemgent), and 3 µM CHIR99021 (Stemgent). mESCs were maintained at 37°C and 5% CO2 and passaged every 2 days.

extract_protocol:
The genomic DNA was prepared by cell harvesting with centrifugation for 5 min at 1000 x g and room temperature, and DNA extraction with Quick-DNA Plus kit (Zymo Research) according to the manufacturer’s protocol.
mESC gDNA was spiked with 0.5% of methylated lambda DNA, 0.025% of 2kb-unmodified and 0.025% of 2kb-caC spike-in controls. For CAPS approach, gDNA was fragmented by Covaris M220 instrument and size-selected to 200-400 bp using Ampure XP beads (Beckman Coulter). For other approaches, gDNA was fragmented and size-selected to 300-500 bp. 0.01% of synthetic oligo with N5mCNN /N5hmCNN sequences and 0.01% of synthetic oligo with 5fC modifications were added after size-selection. 100 ng of fragmented DNA was used for end-repair/A-tailing and ligation of NEBNext Adaptor (NEB) with KAPA Hyper kit (KAPA) according to the manufacturer’s protocol. The uracil in the loop of NEBNext Adaptor was removed by adding 3 μL of USER enzyme (NEB) to the ligation reaction and incubating for 15 min at 37°C. Then the reaction was purified with 0.8X Ampure XP beads according to the manufacturer’s protocol. For CAPS approach, 80% of acetonitrile: H2O was used instead of 80% ethanol: H2O during beads purification step.
OTHER: subtraction-free and bisulfite-free specific sequencing of DNA methylation

value: E14 mouse embryonic stem cells

tag: method

value: Pyridine borane Sequencing (PS) of whole-genome  5-carboxylcytosine (5caC)


title: 320412: CTL_18

value: Male

value: Proximal


title: HH-034

value: Serous Papillary

tag: Stage

value: late


title: HH-033

value: Serous


title: PS

tag: cell type

value: Pyridine borane Sequencing (PS) of whole-genome 5-formylcytosine (5fC)


title: slice_1d_rep1: brain-slice-microglia-1d-E7

value: 10X Genomics 3' Gene Expression Kit v2


title: 320413: EtOH_18

value: Colon Organoid

tag: gender

value: EtOH

value: RNA-sequencing


title: HH-032

value: Serous adenocarcinoma


title: 320411: EtOH_19

value: Female

tag: treatment

value: Distal


title: in_vitro_rep2: 2D-in-vitro-culture-microglia-3wks-F1

source_name: in vitro cultured microglia

tag: tissue

value: in vitro cultured microglia

value: in vitro mixed glial culture after 3 weeks


title: CAPS

value: E14 mouse embryonic stem cells

value: CAPS (Chemical-Assisted Pyridine borane Sequencing)


title: control fed rep3

description:
all_comparisons.txt
S 3

value: wild type

value: whole larvae

value: 3rd instar larvae


title: HH-031

value: Serous Papillary

value: late


title: 320410: CTL_19

value: CTL

tag: sample type


title: TAPSbeta

value: TET assisted pyridine borane sequencing


title: in_vitro_rep1: 2D-in-vitro-culture-microglia-3wks-E1

value: CD11b high; CD45 intermediate

value: 10X Genomics 3' Gene Expression Kit v2


title: 320409: EtOH_20

value: Colon Organoid

value: Male

value: EtOH

value: Proximal

value: RNA-sequencing


title: adult_rep1: Naive-Ex-vivo-microglia

value: microglia

value: brain tissue, adult


title: TLSMGCL 120h

city: Berlin

first: Helene

last: Kretzmer

address: Ihnestraße 63

department: Genome Regulation

country: Germany

institute: Max Planck Institute for Molecular Genetics

description: Single-cell RNA-sequencing of 3 pooled TLSMGCL (at 120h)

data_processing:
Raw base call (BCL) files generated by Illumina sequencers were demultiplexed into FASTQ files using 10X Genomics' Singel-Cell Gene Expression Software Cell Ranger ("mkfastq" pipeline, version 1.3.1, default parameters).
For each experiment FASTQ files were aligned, filtered, barcode and UMI counted using 10X Genomics' Singel-Cell Gene Expression Software Cell Ranger ("count pipeline", version 1.3.1, default parameters). The count pipeline was run jointly on multiple sequencing runs of the same experiment. Genomic reference: "refdata-cellranger-mm10-1.2.0", prepared by 10x Genomics.
The output of Cell Ranger "count" was aggregated and normalized to the same sequencing depth and then the gene-barcode matrices were recomputed on the combined data. Sample aggregation was evaluated with normalizing for sequencing depth differences in the libraries. Normalized UMI counts used the 'mapped' normalization mode. Analysis of the data was based on the normalized UMI counts.
Genome_build: mm10
Supplementary_files_format_and_content: For all samples there is one tar gz archive containing the gene-barcode matrices as produced by the Cell Ranger pipeline. They contain the MEX-formatted UMI count matrices of each sample, or if multiple libraries where sequence, the aggregated gene-barcode matrix. 10x Genomics explains their MEX-formatted UMI counts as follows: "The cellranger pipeline generates a gene-barcode matrix... Each matrix is stored in Market Exchange Format (MEX). It also contains TSV files with genes and barcode sequences corresponding to row and column indices, respectively." More information on the formatting can be found at https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/output/matrices.

source_name: TLSMGCL 120h aa (3x pooled)

growth_protocol:
mESCs culture: Mouse embryonic stem cells (mESCs)  were cultured on 6 cm plates gelatinized with 0.1% gelatin and coated with feeder cells (3-4x10^4cells/cm²)  in ES+LIF medium at 37 °C and 5%  or 7.5%  CO2. mESCs were split every second day with a dilution suitable to the proliferation velocity (between 1/5 and 1/9). ES+LIF medium was refreshed daily. For splitting, media was aspirated and cells were washed once with PBS and trypsinized for 5-10’ at 37 °C. Trypsin was neutralized by 3 ml ES+LIF and centrifuged for 5’ at 1000 rpm, after which the pellet was resuspended in ES+LIF. For freezing of mESCs, cell pellets were resuspended in ES medium with 20% FCS, and mixed in a 1:1 ratio with ES freezing medium. Cells were frozen down o/n in the –80 °C and transferred to liquid nitrogen the next day.
Generation of gastruloids: Gastruloids were generated as described previously (Beccari et al., Nature 2018), with some minor modifications. First, mESCs were feeder freed. To this end, mESCs were trypsinized on the feeder plate as described above, washed with ES+LIF and resuspended in 2ml ES+LIF. On three gelatinized (0.1% gelatin) wells of a 6-well plate, cells were sequentially plated for 25’, 20’ and 15’. With each transfer, cells were triturated to maintain a single cell suspension. Feeder-freed mESCs were then washed once in 5 ml PBS containing MgCl2 and NaCl and once in 5 ml NDiff227. mESCs were then pelleted by centrifugation for 5’ at 1000rpm and resuspended in 500 µl of N2B27. 10 µl of the cell suspension was mixed with 10 µl of Trypanblue (Bio-Rad) for automated cell counting with Luna Automated Cell Counter. 200-250 live cells were then  plated in a volume of 30 to 40 µl NDiff227 into each well of a 96-well round bottom, low attachment plate  (Cellstar 96 well suspension culture plate or Costar 7007 ultra-low attachment 96 well plate). Cells were then allowed to aggregate for 48 h. After these 48h cells were pulsed with 3 µM CHIR99021 (CHI) in 150 µl of  NDiff227. Between 72 and 120h aa, medium was refreshed every 24 h by removing 150 µl of the old media and adding the same volume of new, pre-incubated NDiff227. For the "96h" samples cells harvested at 96h aa and for the "Gastruloid" at 120h aa and subjected to bulk RNA sequencing and for 96h also for single cell RNAseq ("TLS precursor").
Generation of TLS: The gastruloid protocol described above was followed until 96h aa. Gastruloids were then embedded in 5% Growth-Factor-Reduced Matrigel (MG) (Corning). To this end, fresh NDiff227 medium was pre-incubated for at least 20’ at at 37 °C and 5%  or 7.5%  CO2. Pre-incubated medium was then put on ice for 5’, after which MG was added to achieve a final concentration of 5%. Medium was then put on room temperature for 5’, during which 150µl of old medium was removed from the aggregates. New medium with 5%MG (150µl) was then added, and the cultures were returned to the incubator and further cultured at 37 °C and 5%  or 7.5%. Cultured were allowed to settle for at least 30’ before proceeding to further experimentation (e.g. live imaging). For TLS treated with CHI ("TLSMGC") and CHI+LDN ("TLSMGCL"), 5 µM CHI with or without 600 nM LDN was added from 96h to 120h aa prior to adding the MG. For controls ("TLSMG"), an equal volume of diluent (DMSO) was added. All conditions were subjected to bulk RNAseq and the TLSMG were subjected to single cell RNAseq at 108h and 120h aa.

extract_protocol:
Structures were serially washed through pipette transferring (cut 200ul tip) in wells filled with 200ul of 1xPBS/0.4%BSA (5 transfers) to get rid of the Matrigel embedding. Then structures of the same time point have been pooled together and dissociated in a drop of 200 µl TrypLE Express (Gibco) for 20 minutes with pipetting every 5 minute intervals. The cell suspension was filtered using Scienceware Flowmi Cell Strainers, 40 µM. Cells were washed twice with 1 ml 1xPBS/0.4%BSA with centrifugation steps performed for 5 minutes at 1200 rpm in low DNAbind Eppendorf tubes. The cell concentration was determined using a hemocytometer and cells were subjected to single-cell RNA sequencing (10x Genomic, Chromium™ Single Cell 3’ v3; one reaction per timepoint has been used) aiming for a target cell recovery of up to 10,000 sequenced cells per sequencing library (timepoint).
Single-cell libraries were generated following the manual instructions of 10x Genomic (Chromium™ Single Cell 3’ v3), with the exception of fewer PCR cycles in cDNA amplification and sample index PCR than recommended in order to increase complexity of libraries. Libraries were sequenced with a minimum of 230 Mio paired end reads and parameters as described in the manual.

value: TLSMGCL 120h aa (3x pooled)

value: mESC

tag: genotype


title: HH-035

value: Serous

tag: grade

value: high


title: HH-030

value: Serous Papillary


title: 320408: CTL_20


title: HH-029

value: late


title: HH-028


title: slice_3wks_rep1: Invitro-Slice-Culture-microglia

value: brain slice microglia

tag: tissue origin

value: cultured brain slice after 3 weeks


title: Gastruloid 120h

description: Single-cell RNA-sequencing of 5 pooled Gastruloids (at 120h)

source_name: Gastruloid 120h aa (5x pooled)

extract_protocol:
Structures were serially washed through pipette transferring (cut 200ul tip) in wells filled with 200ul of 1xPBS/0.4%BSA (5 transfers). Then structures of the same time point have been pooled together and dissociated in a drop of 200 µl TrypLE Express (Gibco) for 15 minutes with pipetting every 5 minute intervals. The cell suspension was filtered using Scienceware Flowmi Cell Strainers, 40 µM. Cells were washed twice with 1 ml 1xPBS/0.4%BSA with centrifugation steps performed for 5 minutes at 1200 rpm in low DNAbind Eppendorf tubes. The cell concentration was determined using a hemocytometer and cells were subjected to single-cell RNA sequencing (10x Genomic, Chromium™ Single Cell 3’ v3; one reaction per timepoint has been used) aiming for a target cell recovery of up to 10,000 sequenced cells per sequencing library (timepoint).
Single-cell libraries were generated following the manual instructions of 10x Genomic (Chromium™ Single Cell 3’ v3), with the exception of fewer PCR cycles in cDNA amplification and sample index PCR than recommended in order to increase complexity of libraries. Libraries were sequenced with a minimum of 230 Mio paired end reads and parameters as described in the manual.

value: Gastruloid 120h aa (5x pooled)

tag: strain


title: 320407: EtOH_21


title: WT-PBS-6: in vivo PBS control microglia_rep4

data_processing:
Analysis was carried out with Omicsoft ArrayStudio V10.1.1.6 as below
Reads were aligned to the reference with the ArrayStudio V10.1.1.6 Omicsoft Sequence Aligner command "MapRnaSeqReadsToGenome" with parameters Trimming  /Mode=TrimByQuality /ReadTrimQuality=2; /ParallelJobNumber=99 /PairedEnd=True /FileFormat=AUTO /AutoPenalty=True /FixedPenalty=2 /Greedy=false /IndelPenalty=2 /DetectIndels=True /MaxMiddleInsertionSize=10 /MaxMiddleDeletionSize=10 /MaxEndInsertionSize=10 /MaxEndDeletionSize=10 /MinDistalEndSize=3 /ExcludeNonUniqueMapping=False /ReportCutoff=10 /ThreadNumber=4 /InsertSizeStandardDeviation=40 /ExpectedInsertSize=300 /MatePair=False /InsertOnSameStrand=False /InsertOnDifferentStrand=True /QualityEncoding=Automatic /CompressionMethod=Gzip /Gzip=True /SearchNovelExonJunction=True /ExcludeUnmappedInBam=False /KeepFullRead=False /Replace=False /Platform=ILLUMINA /CompressBam=False;
Reads were assigned to genes with Omicsoft command "ReportGeneTranscriptCounts" with parameters /ExpressionMeasurement=RPKM+Count /ParallelJobNumber=1 /Add1=False /CountFragments=True /ExcludeMultiReads=False /Tpm=False /Rpm=False /CountStrandedReads=True /CountReverseStrandedReads=True /AutoTrimUtr=False;
Genome_build: mm10
Supplementary_files_format_and_content: tab-separated gene counts table

source_name: microglia, isolated from mice 10-15 weeks of age

extract_protocol:
Microglia were isolated under ice cold conditions. Pure populations of microglia were obtained by cell sorting for live CD11b positive, CD45 intermediate cells. Sorted cells were collected directly into ice cold lysis buffer and RNA was isolated using the Qiagen RNeasy Micro RNA Kit. Flash frozen brain tissue samples were homogenized and RNA was isolated using the Qiagen RNeasy Mini RNA kit.
Takara SMART-Seq v4 Ultra Low Input RNA Kit + Illumina Nextera XT Library Prep Kit

value: PBS control, 24 hours

tag: immunophenotype

value: CD11b high; CD45 intermediate


title: TLSMGCL 120h rep3

description: Bulk RNA-sequencing of 6 pooled TLSMGCL (gastruloids at 120h in matrigel and CHIR and LDN)

data_processing:
RNAseq reads were pre-processed using cutadapt v2.4 to remove adapter and trim low quality bases.
Reads were subsequently aligned against the reference gnenome mm10 using STAR v2.7.3a (parameter: outSAMtype BAM SortedByCoordinate --outSAMattributes Standard --outSAMstrandField intronMotif --outSAMunmapped Within --quantMode GeneCounts).
Stringtie v2.0 was used for transcript quantification, e.g. calculation of strand-specific coverage, RPKMs and TPMs (reference: mm10 gencode gene annotation gtf).
genome build: mm10
Tab-delimited file containing gene abundances: <Gene ID> <Gene Name> <Reference> <Strand> <Start> <End> <Coverage> <RPKM> <TPM>

source_name: TLSMGCL (6x pooled)

extract_protocol:
Structures have been generated as described below. The experiment has been performed in 3 biological replicates . 6 structures per replicate have been selected (based on the presence of an asymmetric pole) and pooled in a tube. All samples have been washed twice with 1xPBS/0.4%BSA. Then 350ul of RLT Plus buffer containing 1% β-mercaptoethanol (Thermo Cat. No. 21985023) have been added to dissociate the structures and lysate the cells. After pipette dissociation and vortexing, samples have been frozen at -80C. The following day, RNA has been extracted using RNeasy Plus Micro Kit (Qiagen Cat No./ID: 74034) and RNA concentration and quality has been established using Agilent RNA 6000 Pico kit (5067-1513) on an Agilent 2100 Bioanalyzer. All samples analyzed had a RINe values above 8.0, and were then used for library prep.
mRNA libraries have been prepared using KAPA Stranded RNA-Seq Kit (KapaBiosystem 07962142001) following the standard manual protocol. 500ngs of total RNA have been used for each sample to enter the library preparation protocol. For adapter ligation dual indexes have been used (NEXTFLEX® Unique Dual Index Barcodes NOVA-514150) at a working concentration of 71nM (5ul of 1uM stock in each 70ul ligation reaction). Quality and concentration of the obtained libraries have been measured using Agilent High Sensitivity D5000 ScreenTape (5067-5588) on an Agilent 4150 TapeStation. All libraries have been sequenced using 75bp-paired end sequencing (150 cycles kit; FC-410-1002) in a HiSeq4000 platform at a minimum of 23,7 million fragments per sample.


title: WT-PBS-2: in vivo PBS control microglia_rep2

tag: study id

value: Takara SMART-Seq v4 Ultra Low Input RNA Kit + Illumina Nextera XT Library Prep Kit


title: HH-027


title: WT-PBS-4: in vivo PBS control microglia_rep3

tag: rna-seq protocol


title: TLSMGCL 120h rep2

value: 6 pooled TLSMGCL (gastruloids at 120h in matrigel and CHIR and LDN)


title: HH-026

tag: pfs


title: HH-025

tag: histology


title: 320405: EtOH_22

value: Colon Organoid

value: Female

value: EtOH

tag: age

value: Distal


title: 320406: CTL_21

value: Male

value: CTL

value: Proximal

value: RNA-sequencing


title: WT-PBS-10: in vivo PBS control microglia_rep1

value: microglia


title: 320404: CTL_22


title: TLSMGCL 120h rep1

value: mESC


title: TLSMGC 120h rep3

description: Bulk RNA-sequencing of 6 pooled TLSMGC (gastruloids at 120h in matrigel and CHIR)

source_name: TLSMGC (6x pooled)


title: WT-LPS-9: in vivo LPS microglia_rep4

value: LPS 1 mg/kg, 24 hours


title: HH-024

value: Serous

value: late


title: TLSMGC 120h rep2

value: 6 pooled TLSMGC (gastruloids at 120h in matrigel and CHIR)


title: WT-LPS-5: in vivo LPS microglia_rep3

value: CD11b high; CD45 intermediate


title: 320402: CTL_23


title: HH-023

value: Serous Papillary


title: 320401: EtOH_24


title: TLSMGC 120h rep1


title: WT-LPS-3: in vivo LPS microglia_rep2

value: Takara SMART-Seq v4 Ultra Low Input RNA Kit + Illumina Nextera XT Library Prep Kit


title: HH-022


title: TLSMG 120h rep3

description: Bulk RNA-sequencing of 6 pooled TLSMG (gastruloids at 120h in matrigel)

source_name: TLSMG (6x pooled)


title: HH-021


title: WT-LPS-1: in vivo LPS microglia_rep1


title: 320400: CTL_24

value: Colon Organoid


title: TLSMG 120h rep2

value: 6 pooled TLSMG (gastruloids at 120h in matrigel)


title: WT-LPS24h-9: brain tissue LPS_rep4

data_processing:
Analysis was carried out with Omicsoft ArrayStudio V10.1.1.6 as below
Reads were aligned to the reference with the Omicsoft Sequence Aligner command "MapRnaSeqReadsToGenome" with parameters Trimming  /Mode=TrimByQuality /ReadTrimQuality=2; Options  /ParallelJobNumber=99 /PairedEnd=False /FileFormat=AUTO /AutoPenalty=True /FixedPenalty=2 /Greedy=false /IndelPenalty=2 /DetectIndels=True /MaxMiddleInsertionSize=10 /MaxMiddleDeletionSize=10 /MaxEndInsertionSize=10 /MaxEndDeletionSize=10 /MinDistalEndSize=3 /ExcludeNonUniqueMapping=False /ReportCutoff=10 /ThreadNumber=8 /InsertSizeStandardDeviation=40 /ExpectedInsertSize=300 /MatePair=False /InsertOnSameStrand=False /InsertOnDifferentStrand=True /QualityEncoding=Automatic /CompressionMethod=Gzip /Gzip=True /SearchNovelExonJunction=True /ExcludeUnmappedInBam=False /KeepFullRead=False /Replace=False /Platform=ILLUMINA /CompressBam=False;
Reads were assigned to genes with Omicsoft command "ReportGeneTranscriptCounts" with parameters /ExpressionMeasurement=RPKM+Count_Gene+Transcript /ParallelJobNumber=99 /Add1=False /CountFragments=True /ExcludeMultiReads=False /Tpm=True /Rpm=False /CountStrandedReads=True /CountReverseStrandedReads=True /AutoTrimUtr=False;
Genome_build: mm10
Supplementary_files_format_and_content: tab-separated gene counts table

source_name: brain tissue, isolated from mice 10-15 weeks of age

extract_protocol:
Microglia were isolated under ice cold conditions. Pure populations of microglia were obtained by cell sorting for live CD11b positive, CD45 intermediate cells. Sorted cells were collected directly into ice cold lysis buffer and RNA was isolated using the Qiagen RNeasy Micro RNA Kit. Flash frozen brain tissue samples were homogenized and RNA was isolated using the Qiagen RNeasy Mini RNA kit.
Illumina TruSeq mRNA Library Prep Kit

value: brain tissue

value: not applicable

value: Illumina TruSeq mRNA Library Prep Kit


title: HH-020


title: 320403: EtOH_23

value: Male

value: EtOH

value: Distal


title: WT-LPS24h-12: brain tissue LPS_rep3


title: Gastruloid rep3

description: Bulk RNA-sequencing of 6 pooled gastruloids at 120h (Beccari protocol)

source_name: Gastruloid (6x pooled)


title: HH-019

value: Low grade serous carcinoma

value: low


title: HH-017

value: high


title: HH-018

value: papillary serous adenocarcinoma


title: 320398: CTL_25

value: Female

value: CTL

value: Proximal

value: RNA-sequencing


title: 320397: EtOH_26


title: Gastruloid rep2

value: mESC

value: 6 pooled gastruloids at 120h (Beccari protocol)


title: 320396: CTL_26

value: Colon Organoid


title: Gastruloid rep1


title: 320395: EtOH_27


title: WT-UT-4: brain tissue untreated LPS control_rep4

value: brain tissue

value: no treatment


title: WT-LPS24h-11: brain tissue LPS_rep2

value: LPS 1 mg/kg, 24 hours

value: not applicable

value: Illumina TruSeq mRNA Library Prep Kit


title: 96h rep3

description: Bulk RNA-sequencing of 10 pooled TLS precursor at 96h

source_name: 96h aa (10x pooled)

extract_protocol:
Structures have been generated as described below. The experiment has been performed in 3 biological replicates . 10 structures per replicate have been selected (based on the presence of an asymmetric pole) and pooled in a tube. All samples have been washed twice with 1xPBS/0.4%BSA. Then 350ul of RLT Plus buffer containing 1% β-mercaptoethanol (Thermo Cat. No. 21985023) have been added to dissociate the structures and lysate the cells. After pipette dissociation and vortexing, samples have been frozen at -80C. The following day, RNA has been extracted using RNeasy Plus Micro Kit (Qiagen Cat No./ID: 74034) and RNA concentration and quality has been established using Agilent RNA 6000 Pico kit (5067-1513) on an Agilent 2100 Bioanalyzer. All samples analyzed had a RINe values above 8.0, and were then used for library prep.
mRNA libraries have been prepared using KAPA Stranded RNA-Seq Kit (KapaBiosystem 07962142001) following the standard manual protocol. 500ngs of total RNA have been used for each sample to enter the library preparation protocol. For adapter ligation dual indexes have been used (NEXTFLEX® Unique Dual Index Barcodes NOVA-514150) at a working concentration of 71nM (5ul of 1uM stock in each 70ul ligation reaction). Quality and concentration of the obtained libraries have been measured using Agilent High Sensitivity D5000 ScreenTape (5067-5588) on an Agilent 4150 TapeStation. All libraries have been sequenced using 75bp-paired end sequencing (150 cycles kit; FC-410-1002) in a HiSeq4000 platform at a minimum of 23,7 million fragments per sample.

value: 10 pooled TLS precursor at 96h


title: WT-LPS24h-10: brain tissue LPS_rep1


title: HH-016

source_name: Ovarian tumour

value: Serous

value: late


title: HH-015

value: low


title: WT-UT-3: brain tissue untreated LPS control_rep3


title: HH-014

value: high


title: 320394: CTL_27

source_name: Colon Organoid

value: Female


title: 320399: EtOH_25

value: EtOH


title: TLSMG 120h rep1

value: 6 pooled TLSMG (gastruloids at 120h in matrigel)


title: WT-UT-2: brain tissue untreated LPS control_rep2


title: 96h rep1


title: WT-UT-1: brain tissue untreated LPS control_rep1


title: 320392: CTL_28

value: CTL

value: Distal

value: RNA-sequencing


title: TLSMG 108h

description: Single-cell RNA-sequencing of 5 pooled TLSMG (at 108h)

data_processing:
Raw base call (BCL) files generated by Illumina sequencers were demultiplexed into FASTQ files using 10X Genomics' Singel-Cell Gene Expression Software Cell Ranger ("mkfastq" pipeline, version 1.3.1, default parameters).
For each experiment FASTQ files were aligned, filtered, barcode and UMI counted using 10X Genomics' Singel-Cell Gene Expression Software Cell Ranger ("count pipeline", version 1.3.1, default parameters). The count pipeline was run jointly on multiple sequencing runs of the same experiment. Genomic reference: "refdata-cellranger-mm10-1.2.0", prepared by 10x Genomics.
The output of Cell Ranger "count" was aggregated and normalized to the same sequencing depth and then the gene-barcode matrices were recomputed on the combined data. Sample aggregation was evaluated with normalizing for sequencing depth differences in the libraries. Normalized UMI counts used the 'mapped' normalization mode. Analysis of the data was based on the normalized UMI counts.
genome build: mm10
For all samples there is one tar gz archive containing the gene-barcode matrices as produced by the Cell Ranger pipeline. They contain the MEX-formatted UMI count matrices of each sample, or if multiple libraries where sequence, the aggregated gene-barcode matrix. 10x Genomics explains their MEX-formatted UMI counts as follows: "The cellranger pipeline generates a gene-barcode matrix... Each matrix is stored in Market Exchange Format (MEX). It also contains TSV files with genes and barcode sequences corresponding to row and column indices, respectively." More information on the formatting can be found at https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/output/matrices.

source_name: TLSMG 108h aa (5x pooled)

value: mESC

value: 5 pooled TLSMG (at 108h)


title: 320391: EtOH_29

value: Colon Organoid

value: Proximal


title: HH-012


title: Hmga2-/- Gadd45a

city: Nancy Cedex

first: Guillermo

last: Barreto

address: 9, Avenue de la Forêt de Haye ‐ BP 20199

department: Campus Biologie-Santé. Faculté de Médecine

country: France

institute: CNRS Director of Research

data_processing:
Visualization of raw reads was done using FastQC. Raw reads of replicates were merged and trimmed using Trimmomatic (Bolger et al. 2014), using the following parameters LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:50 CROP:63 HEADCROP:13
High quality reads were aligned to the mm10 genome by using bowtie2 with default parameters.
Sam files were converted to bam files using samtools view and sort.
Bigwig files were generated using deeptool's bamCompare (--operation log2  --normalizeUsing RPKM). A Bed-File containing UCSC known genes were downloaded using the Table Browser from UCSC Genome Browser and used as region-file.
Genome_build: mm10
Supplementary_files_format_and_content: BigWig files were generated using deeptool's bamCompare; Scores represent compared values.

source_name: Mouse embryonic fibroblasts

growth_protocol: MEF cells were kept at 37°C in DMEM medium containing 10 % Fetal Calf Serum, and 1% P/S/G (penicillin/Streptomycin/L-glutamine)

extract_protocol:
Cells were cross-linked with 1% formaldehyde for 10 min at room temperature. The reaction was quenched for 5 min on ice by adding 0.125 M glycine. After three washes with 1x PBS, cells were subjected to hypotonic buffer L1 (50 mM Tris-HCl pH 8.0, 2 mM EDTA pH 8.0, 0.1% IGEPAL CA-630 (Sigma), 10% glycerol, 2 mM DTT, 10 mM NaF, 2 mM Na3VO4, 0.2 M imidazole, 1 mM PMSF and protease inhibitor cocktail). Cells were lysed, and sonicated with Diagenode Bioruptor to an average DNA length of 100-300 bp. After centrifugation, the soluble chromatin was immunoprecipitated using antibodies specific against H3 (Abcam), H2A.X (Millipore), pPolII (Abcam) and GADD45A (Santacruz). Precipitated chromatin complexes were removed from the beads and decrosslinked by incubating with 100 μl of 10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 0.1 M NaHCO3, 1% SDS and Proteinase K at 65 °C overnight. Reverse cross-linked immunoprecipitated chromatin was purified using the QIAquick PCR purification kit (Qiagen).
Libraries were prepared according to Illumina's instructions accompanying the Ovation Ultra Low Kit.

value: Mouse embryonic fibroblasts

value: whole embryo

value: embryonic day 15.5

value: Hmga-/-

tag: cell extraction

value: Timed-pregnant mice were sacrificed on post coitum day 15.5  according to standard methods. The embryos were collected in sterile PBS complemented with 100 U\ml penicillin and 100 U\ml streptomycin. After removing the head, limbs, tail, viscera, liver and heart, the remaining body was trypsinized with 1x trypsin-EDTA solution (0.05 %) for 20 minutes at 37°C and single cells were obtained via physical disruption by sterile Pasteur pipet. The cells from each embryo were diluted with growth medium containing FCS and plated separately.

tag: antibody

value: Gadd45a(Santacruz, sc-797)


title: HH-011

value: Serous

value: high

tag: Stage

value: late


title: 320390: CTL_29

tag: cell type

tag: gender

value: Female


title: 96h

description: Single-cell RNA-sequencing of 6 pooled TLS precursor (at 96h)

source_name: 96h aa (6x pooled)

tag: tissue

value: 6 pooled TLS precursor (at 96h)


title: Hmga2-/- pPolII

value: serine 5 phosphorylated RNA polymerase II (Abcam, ab5408)


title: 320389: EtOH_30

value: Male

tag: treatment

value: EtOH

tag: sample type

value: RNA-sequencing


title: dme_Piwi_IP_WT_ov_1

city: Baltimore

first: Manolis

last: Maragkakis

state: MD

address: 251 Bayview Blvd

institute: National Institute on Aging

data_processing:
Adaptor trimming: The adaptor was trimmed with cutadapt v2.3
Remove duplicates (only for libraries with 8 random nucleotides at the end of reads): Remove duplicate sequences from the fastq and then trim the 8 nt from the 3' end of reads
Alignment: Reads were aligned using STAR v2.4.2a to the genome (dm3) and to consensus transposon sequences.
Aligned reads were loaded into a SQLite3 database for further processing with CLIPSeqTools and annotated with information whether they overlap with elements from RepeatMasker (downloaded from UCSC) and genes (from UCSC gene model annotation file).
Genome_build: dm3
Supplementary_files_format_and_content: Processed files are TAB delimited files quantifying the number of reads on the sense and antisense strands of the consensus transposon sequences. Files contain the following columns: sample (sample name), category (sense or antisense), ref (transposon_name), count (number of collapsed reads), copyNumber (number of reads) , celltype

source_name: Ovaries

growth_protocol: Fly strains were grown on standard cornmeal molasses at 25°C, with 70% relative humidity in a 12-h light–dark cycle.

extract_protocol: Small RNAs were Trizol extracted from immunopurified PIWI clade proteins isolated from fly ovary lysates.

treatment_protocol: Fly ovaries were dissected in cold PBS from 2-5 day old yeast fed flies.

value: a-Piwi-N7 Mourelatos lab

value: Ovaries


title: Hmga2-/- H3

value: Mouse embryonic fibroblasts

tag: genotype

value: H3 (Abcam, ab1791)


title: HH-010

tag: grade


title: 96h rep2

value: mESC

tag: strain

value: 10 pooled TLS precursor at 96h


title: Hmga2-/- H2AX

value: whole embryo

value: embryonic day 15.5

value: Hmga-/-

value: Timed-pregnant mice were sacrificed on post coitum day 15.5  according to standard methods. The embryos were collected in sterile PBS complemented with 100 U\ml penicillin and 100 U\ml streptomycin. After removing the head, limbs, tail, viscera, liver and heart, the remaining body was trypsinized with 1x trypsin-EDTA solution (0.05 %) for 20 minutes at 37°C and single cells were obtained via physical disruption by sterile Pasteur pipet. The cells from each embryo were diluted with growth medium containing FCS and plated separately.

value: H2AX (Millipore, 07-627)


title: 320388: CTL_30

value: Colon Organoid

value: CTL

value: Proximal


title: 320387: EtOH_31

value: Female

value: Distal


title: Hmga2-/- Input


title: HH-009

value: Serous

value: high

value: late


title: 320393: EtOH_28

value: EtOH

value: RNA-sequencing


title: HH-008


title: HH-013


title: Hmga2+/+ Gadd45a

value: Mouse embryonic fibroblasts

value: embryonic day 15.6

value: wild type

value: Gadd45a(Santacruz, sc-797)


title: HH-006


title: rrp4-G226D_3

city: Atlanta

first: Anita

middle: H

last: Corbett

state: Georgia

address: 1510 Clifton Road

department: Biology

institute: Emory University

data_processing:
Base-calling: Illumina RTA 1.18.64 software
Demultiplexing: Bcl2fastq v2.17 software
Alignment: Reads were mapped to the S. cerevisiae S288C genome assembly R64-1-1, annotated with CUTs and SUTs (Xu et al. Nature. 2009), using the STAR RNA-seq aligner [v2.7.2b (Dobin et al. Bioinformatics. 2012)]
Counting: Reads were assigned to gene features (Gene_ID) and counted using featureCounts [v1.6.4+galaxy1 (Liao et al. Bioinformatics. 2014)]
Differential gene expression analysis: DE analysis on raw read counts was performed using DESeq2 [v2.11.40.6+galaxy1(Love et al. Genome Biology. 2014)]
Genome_build: S. cerevisiae S288C R64-1-1
Supplementary_files_format_and_content: Tabular files contain raw gene counts for each sample produced using featureCounts
Supplementary_files_format_and_content: Tab-delimited text file contains normalized abundance measurements generated using DESeq2

organism: Saccharomyces cerevisiae

source_name: S. cerevisiae cells (haploid)_rrp4-G226D

growth_protocol: S. cerevisiae rrp4∆ cells containing RRP4-Myc or rrp4-G226D-Myc plasmid (pRS315; LEU2; CEN)were grown in 10 mL Leu- minimal media in triplicate at 37°C for 5hr.

extract_protocol:
Total RNA extracted from S. cerevisiae cells using MasterPure™ Yeast RNA Purification Kit (Epicentre, Lucigen)
Stranded cDNA libraries prepared with TruSeq Total RNA Stranded Library Prep kit (Illumina)

tag: genetic information

value: MAT⍺; ura3∆0; leu2∆0; his3∆1; rrp4∆::NEO; [rrp4-G226D-2xMyc; LEU2; CEN; pRS315]


title: rrp4-G226D_2


title: 320385: EtOH_32

value: Male


title: HH-005


title: Hmga2+/+ pPolII

value: whole embryo

value: embryonic day 15.5

value: Timed-pregnant mice were sacrificed on post coitum day 15.5  according to standard methods. The embryos were collected in sterile PBS complemented with 100 U\ml penicillin and 100 U\ml streptomycin. After removing the head, limbs, tail, viscera, liver and heart, the remaining body was trypsinized with 1x trypsin-EDTA solution (0.05 %) for 20 minutes at 37°C and single cells were obtained via physical disruption by sterile Pasteur pipet. The cells from each embryo were diluted with growth medium containing FCS and plated separately.

value: serine 5 phosphorylated RNA polymerase II (Abcam, ab5408)


title: rrp4-G226D_1


title: 320384: CTL_32

value: Colon Organoid

value: CTL

value: Proximal


title: dme_Piwi_IP_RK_ov_2

value: a-Piwi-N7 Mourelatos lab

value: Ovaries


title: Hmga2+/+ H3

value: H3 (Abcam, ab1791)


title: dme_Ago3_IP_WT_ov_1

value: aAgo3-380 Mourelatos lab


title: RRP4_3

source_name: S. cerevisiae cells (haploid)_RRP4

value: MAT⍺; ura3∆0; leu2∆0; his3∆1; rrp4∆::NEO; [RRP4-2xMyc; LEU2; CEN; pRS315]


title: HH-004

value: Serous

value: high

value: late


title: Hmga2+/+ H2AX

value: Mouse embryonic fibroblasts

value: wild type

value: H2AX (Millipore, 07-627)


title: RRP4_2


title: 320383: EtOH_33

value: Female

value: EtOH

value: Distal

value: RNA-sequencing


title: 320382: CTL_33

value: Colon Organoid

value: CTL


title: Hmga2+/+ Input

value: whole embryo

value: embryonic day 15.5

value: Timed-pregnant mice were sacrificed on post coitum day 15.5  according to standard methods. The embryos were collected in sterile PBS complemented with 100 U\ml penicillin and 100 U\ml streptomycin. After removing the head, limbs, tail, viscera, liver and heart, the remaining body was trypsinized with 1x trypsin-EDTA solution (0.05 %) for 20 minutes at 37°C and single cells were obtained via physical disruption by sterile Pasteur pipet. The cells from each embryo were diluted with growth medium containing FCS and plated separately.


title: RRP4_1


title: HH-003

value: early


title: HH-007


title: 320386: CTL_31


title: HH-002


title: 3W_R2_input

status: Public on Dec 13 2020

city: GuangZhou

first: Yan

last: Li

state: GuangDong

address: No. 135, Xingang Xi Road, Guangzhou, 510275, P. R. China

department: School of Life Science

country: China

institute: Sun Yat-sen University

description: Input sample

data_processing:
RNA-seq and m6A-seq reads were aligned to the genome assembly of Apis mellifera (Amel_4.5) using HISAT (version 2.1.0)
m6A enriched region were detected by exomePeak R package. RNA-Seq data(no IP, input RNA) in the same condition was used as input information
Genome_build: Amel_4.5
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each sample

organism: Apis mellifera

source_name: worker larvae

growth_protocol: The honeybee, Apis mellifera, colonies were maintained at the Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing, China, according to the standard beekeeping technique.

extract_protocol:
Total RNA from each sample was extracted using the RNA-extraction-system with TRIzol Reagent (15596018, Invitrogen). Polyadenylated RNA (polyA-RNA) were extracted using Dynabeads mRNA Purification Kit (61006, Invitrogen)
Immunoprecipitated RNA fragments and comparable amounts of fragmented polyA-RNA (as input) were subjected to library construction with NEBNext Ultra RNA Library Prep Kit for Illumina (E7530S, New England Biolabs) according to the manufacturer’s instructions.

treatment_protocol: we collected five larval samples at three larval development stages in both queen and worker castes: the first-instar larvae (L1,  termed as 1D), the third-instar queen and worker larvae (L3,  termed as 3Q and 3W, respectively), and the fifth-instar queen and worker larvae (L5,termed as 5Q and 5W, respectively).  All tissues were freshly frozen on dry ice and stored at –80°C.

value: larvae

tag: developmental stage

value: 3 instar

tag: bee type

value: worker honeybee

tag: rip antibody


title: 5Q_R1_input

description: Input sample

source_name: queen larvae

value: 5 instar

value: queen honeybee


title: 5W_R1_input

description: Input sample


title: 5Q_R2_input

description: Input sample


title: 5W_R2_input

description: Input sample


title: HH-001

tag: pfs

tag: histology

value: Serous

value: high

value: late


title: 3Q_R2_input

description: Input sample


title: 3Q_R1_input

description: Input sample


title: 3W_R1_input

description: Input sample


title: 1D_R2_input

description: Input sample

source_name: honeybee larvae

value: 1 instar

value: honeybee (this stage larvae can develop into worker larvae or queen larvae)


title: 1D_R1_input

description: Input sample


title: 5W_R2_IP

description: IP sample

value: m6A antibody (Synaptic Systems, catalog# 202003)


title: 5Q_R2_IP

description: IP sample


title: 5W_R1_IP

description: IP sample


title: 3W_R2_IP

description: IP sample


title: 3W_R1_IP

description: IP sample


title: 3Q_R2_IP

description: IP sample


title: 1D_R1_IP

description: IP sample


title: 1D_R2_IP

description: IP sample


title: Rat3_LPS_7h_rep2

city: Edinburgh

first: Clare

last: Pridans

address: 47 Little France Crescent

department: University of Edinburgh

institute: Centre for Inflammation Research

description:
Rat 3 BMDM LPS 7h technical replicate 2
Gene expression data from adult male BMDM cultured in LPS for 7 hours

hyb_protocol: GeneTitan

scan_protocol: GeneChip Command Console

data_processing:
CEL files were normalised (RMA) in R/Bioconductor.
antilog2 RMA signal

label: Biotin

organism: Rattus norvegicus

source_name: Dark agouti Rat 3

label_protocol: GeneChip Whole Transcript Labeling Kit (Applied Biosystems)

growth_protocol: Rat bone marrow and PBMC were cultured in CSF1 for 7 days to produce macrophages. ESDM were derived from ESC grown in IL3 and CSF1, followed by CSF1 alone. EB were harvested when cultured in CSF1 and producing ESDM. AM and PM were cultured overnight in CSF1 following isolation.

extract_protocol: Trizol extraction of total RNA followed by purification with Rneasy Mini kit

treatment_protocol: BMDM were cultured in LPS and RNA isolated at 2, 7 and 24 hours.

value: bone marrow-derived macrophage


title: Rat2_LPS_24h_rep2

description:
Rat 2 BMDM LPS 24h technical replicate 2
Gene expression data from adult male BMDM cultured in LPS for 24 hours

source_name: Dark agouti Rat 2


title: Rat2_LPS_24h_rep1

description:
Rat 2 BMDM LPS 24h technical replicate 1
Gene expression data from adult male BMDM cultured in LPS for 24 hours


title: Rat3_LPS_7h_rep3

description:
Rat 3 BMDM LPS 7h technical replicate 3
Gene expression data from adult male BMDM cultured in LPS for 7 hours


title: Rat2_LPS_7h_rep2

description:
Rat 2 BMDM LPS 7h technical replicate 2
Gene expression data from adult male BMDM cultured in LPS for 7 hours


title: Rat3_LPS_24h_rep1

description:
Rat 3 BMDM LPS 24h technical replicate 1
Gene expression data from adult male BMDM cultured in LPS for 24 hours


title: Rat1_LPS_24h_rep2

description:
Rat 1 BMDM LPS 24h technical replicate 2
Gene expression data from adult male BMDM cultured in LPS for 24 hours

source_name: Dark agouti Rat 1


title: Rat3_LPS_24h_rep2

description:
Rat 3 BMDM LPS 24h technical replicate 2
Gene expression data from adult male BMDM cultured in LPS for 24 hours


title: Rat1_LPS_7h_rep2

description:
Rat 1 BMDM LPS 7h technical replicate 2
Gene expression data from adult male BMDM cultured in LPS for 7 hours


title: Rat1_LPS_7h_rep4

description:
Rat 1 BMDM LPS 7h technical replicate 4
Gene expression data from adult male BMDM cultured in LPS for 7 hours


title: Rat1_LPS_2h_rep2

description:
Rat 1 BMDM LPS 2h technical replicate 2
Gene expression data from adult male BMDM cultured in LPS for 2 hours


title: Rat2_LPS_7h_rep1

description:
Rat 2 BMDM LPS 7h technical replicate 1
Gene expression data from adult male BMDM cultured in LPS for 7 hours


title: Rat2_LPS_2h_rep1

description:
Rat 2 BMDM LPS 2h technical replicate 1
Gene expression data from adult male BMDM cultured in LPS for 2 hours


title: Rat2_LPS_2h_rep2

description:
Rat 2 BMDM LPS 2h technical replicate 2
Gene expression data from adult male BMDM cultured in LPS for 2 hours


title: Rat1_LPS_2h_rep1

description:
Rat 1 BMDM LPS 2h technical replicate 1
Gene expression data from adult male BMDM cultured in LPS for 2 hours


title: Rat3_LPS_2h_rep2

description:
Rat 3 BMDM LPS 2h technical replicate 2
Gene expression data from adult male BMDM cultured in LPS for 2 hours


title: Rat3_LPS_2h_rep1

description:
Rat 3 BMDM LPS 2h technical replicate 1
Gene expression data from adult male BMDM cultured in LPS for 2 hours


title: Liver_G07.KO

description:
Csf1r -/- rat liver
Gene expression data from adult male PM

source_name: Mixed dark agouti/sprague dawley female

value: adult whole liver


title: Liver_C07.KO


title: Liver_A09.KO

source_name: Mixed dark agouti/sprague dawley male


title: Liver_H07.HET

description:
Csf1r +/- rat liver
Gene expression data from adult male PM


title: 3Q_R1_IP

description: IP sample


title: Liver_H05.HET


title: 5Q_R1_IP

description: IP sample


title: Liver_G05.KO


title: Liver_B07.HET


title: Rat1_LPS_24h_rep1

description:
Rat 1 BMDM LPS 24h technical replicate 1
Gene expression data from adult male BMDM cultured in LPS for 24 hours


title: Liver_E07.WT

description:
Wild type rat liver
Gene expression data from adult male PM


title: Liver_D07.WT


title: MDM_Rat3

description:
Monocyte derived macrophages Rat 3
Gene expression data from adult male PM

value: Monocyte derived macrophages


title: MDM_Rat2

description:
Monocyte derived macrophages Rat 2
Gene expression data from adult male PM


title: MDM_Rat1

description:
Monocyte derived macrophages Rat 1
Gene expression data from adult male PM


title: Liver_B09.WT


title: PM_Rat1

description:
Alveolar macrophages Rat 1
Gene expression data from adult male PM

value: Peritoneal macrophages


title: PM_Rat3

description:
Alveolar macrophages Rat 3
Gene expression data from adult male PM


title: Liver_A07.WT


title: AM_Rat3

description:
Alveolar macrophages Rat 3
Gene expression data from adult male AM

value: Alveolar macrophages


title: AM_Rat1

description:
Alveolar macrophages Rat 1
Gene expression data from adult male AM


title: MDM_Rat3_Repeat

description:
Monocyte derived macrophages Rat 3 repeat
Gene expression data from adult male PM


title: PM_Rat2

description:
Alveolar macrophages Rat 2
Gene expression data from adult male PM


title: PM_Rat2_Repeat

description:
Alveolar macrophages Rat 2 repeat
Gene expression data from adult male PM


title: AM_Rat2

description:
Alveolar macrophages Rat 2
Gene expression data from adult male AM


title: Liver_F07.HET


title: Rat2_BMDM_rep2_repeat

description:
Rat 2 BMDM technical replicate 2 repeat
Gene expression data from adult male bone marrow derived macrophages

source_name: Dark agouti Rat 2 BMDM


title: Rat3_BMDM_rep2

description:
Rat 3 BMDM technical replicate 2
Gene expression data from adult male bone marrow derived macrophages

source_name: Dark agouti Rat 3 BMDM


title: DA5.2_ESDM_Rep1_Repeat

description:
ESDM technical replicate 1 repeat
Gene expression data from adult male PM

source_name: Dark Agouti ESDM clone 5.2

value: embryonic stem cell-derived macrophage


title: DA5.2_ESDM_Rep1

description:
ESDM technical replicate 1
Gene expression data from embryonic stem cell derived macrophages


title: Rat2_BMDM_rep1

description:
Rat 2 BMDM technical replicate 1
Gene expression data from adult male bone marrow derived macrophages


title: Rat1_BMDM_rep2

description:
Rat 1 BMDM technical replicate 2
Gene expression data from adult male bone marrow derived macrophages

source_name: Dark agouti Rat 1 BMDM


title: Rat3_BMDM_rep3

description:
Rat 3 BMDM technical replicate 3
Gene expression data from adult male bone marrow derived macrophages


title: Rat1_BMDM_rep3

description:
Rat 1 BMDM technical replicate 3
Gene expression data from adult male bone marrow derived macrophages


title: Rat2_BMDM_rep2

description:
Rat 2 BMDM technical replicate 2
Gene expression data from adult male bone marrow derived macrophages


title: DA5.2_ESDM_Rep3

description:
ESDM technical replicate 3
Gene expression data from embryonic stem cell derived macrophages


title: tSCRB_MM_D8_arm_10

city: Freising

first: Patrick

last: Roelli

address: Weihenstephaner Berg 3

department: Tierphysiologie & Immunologie

institute: TUM

data_processing:
dropSeqPipe 0.4. The pipeline includes filtering mapping and cell/UMI demultiplexing. Each protocols has it's own supplementary configuration file describing all the paramters used.
Filtering is done by cutadapt
Mapping is done by STAR
Demultiplexing is done by drop-seq tools v2
Supplementary_files_format_and_content: custom_adapter.fa - Adapters used in the protocols. Used to trim data in the pipeline
gtf_biotypes.yaml - List of biotypes used for mapping with STAR.
DS_config.yaml - Drop-seq configuration file with all the parameters used in the pipeline
SCRB_config.yaml - scrb-seq configuration file with all the parameters used in the pipeline
SCRB_barcodes csv - List of cell barcodes in the SCRB-seq protocols used in the pipeline
barcodes.tsv.gz - Cell barcodes with prefix
features.tsv.gz - Genes
matrix.mtx.gz - MTX matrix of UMI counts

source_name: tSCRB-seq P14s from day 8 acute infection 10

extract_protocol:
the single-cell transcriptomes are generated with a T-cell tailored version of the SCRB-seq protocol using Qiagen TCL buffer supplemented with Î²-mercaptoethanol for cell lysis and a template switching oligo with locked nucleic acid
[Purification of mouse and human PBMCs]  Mouse and human lymphocytes were isolated from a mouse spleen and human blood with the use of Lympholyte M (Cedarlane) and Ficoll-Paque PLUS (GE Healthcare) density gradient media respectively. Cells were washed with media and sorted on BD FACS Fusion (100-micron nozzle  standard operation settings  single-cell purity)  where individual cells meeting the gating strategy were sorted in a tube containing media and used immediately for generating Drop-seq single-cell transcriptomes.
[Purification of naive P14 T cells]  Single cell splenocyte suspensions were obtained by mashing total spleens through a 100 Î¼m nylon cell strainer (BD Falcon) and lysing red blood cells with a hypotonic ACK buffer. NaÃ¯ve transgenic P14 CD8 T-cells were isolated using the mouse CD8+ T-cell enrichment kit (Miltenyi Biotech  Bergisch-Gladbach  Germany). Surface staining was performed for 40 min at 4Â°C in supplemented DMEM media (Gibco  ThermoFisher Scientific) using the following antibodies: anti-CD8a (53-6.7  eBioscience)  CD4 (RM4-4  Biolegend)  and VÎ±2 (V20.1  eBioscience). Cells were washed twice with media and sorted on BD FACS Fusion (100-micron nozzle  standard operation settings  single-cell purity). Individual cells meeting the gating strategy were sorted in tube containing media (Drop-seq) and used immediately or directly in lysis buffer into individual wells of a low-binding PCR plate (SCRB-seq)  which was subsequently spun down  snap-frozen on dry ice and stored at -80oC until use. All data were analyzed using FlowJo (TreeStar).
[Purification of activated P14 T cells]  Single cell splenocyte suspensions were obtained by mashing total spleens through a 100 Î¼m nylon cell strainer (BD Falcon) and red blood cells were lysed with a hypotonic ACK buffer. NaÃ¯ve transgenic P14 CD8 T-cells were isolated using the mouse CD8+ T-cell enrichment kit (Miltenyi Biotech  Bergisch-Gladbach  Germany). 2x103 CD45.1+ P14 TCRÎ±Î² were transferred into naÃ¯ve CD45.2+ C57BL/6 mice  followed by their infection. On the day of the sort  single cell splenocyte suspensions were obtained as described above. Activated transgenic P14 CD8 T-cells were isolated using anti-CD45.1 biotin/anti-biotin conjugated microbeads and magnetic MACS cell separation (Miltenyi Biotech  Bergisch-Gladbach  Germany). Surface staining was performed for 40 min at 4Â°C in supplemented DMEM media (Gibco  ThermoFisher Scientific) using the following antibodies: anti-CD8a (53-6.7  eBbioscience)  CD4 (RM4-4  Biolegend)  CD45.1 (A20  eBioscience) and CD45.2 (104  eBioscience). Cells were washed twice with media and sorted on BD FACS Fusion (100-micron nozzle  standard operation settings  single-cell purity). Individual cells meeting the gating strategy were sorted in tube containing media (Drop-seq) and used immediately or directly in lysis buffer into individual wells of a low-binding PCR plate (SCRB-seq)  which was subsequently spun down  snap-frozen on dry ice and stored at -80oC until use. All data were analyzed using FlowJo (TreeStar).
[Drop-seq library construction protocol]  The original Drop-seq protocol was performed as described by Macosko and colleagues16. All runs were performed at 3000 Âµl/hour cell and bead rates with the use of Drop-seq PDMS device (Nanoshift). The following reagents were used for the Drop-seq modification described in this article: Lithium dodecyl sulfate (L9781  Sigma-Aldrich)  Lithium chloride (62476  SigmalAldrich)  Igepal (I8896-50ML  Sigma-Aldrich)  NaCl (S3014  Sigma-Aldrich)  3âLNA(G)TSO (IDT)  Maxima H Minus RT (EP0753  ThermoFisher)  SuperScript IV RT (18090010  ThermoFisher)  SMARTScribe RT (639536  Takara Bio) and Ficoll PM 400 (GE17-0300-10  GE Healthcare). After RNA purification and reverse transcription  the generated single-cell cDNA was amplified for 18 cycles. The barcoded single-cell amplicons were purified with the use of (0.6x) Agencourt AMPure XP beads. 1 ng of the resulting amplified cDNA were used for library preparation with the Illumina Nextera XT DNA Library reagents (FC-131-1024  Illumina). After PCR amplification of the fragmented libraries  the samples were purified with (0.6x) Agencourt AMPure XP beads and eluted in 10 Âµl of molecular grade water. The quality of the resulting library was assessed with the use of Agilent High Sensitivity DNA Kit (5067-4626  Agilent). The library quantification was performed based on the Illumina recommendations (SY-930-1010  Illumina) with the use of KAPA SYBR FAST qPCR Master Mix (KK4600  Kapa Biosystems). The samples were sequenced on Illumina HiSeq 2500 system at the following conditions - rapid run  paired-end  20 bp read 1  45 bp read 2  single-indexed sequencing resulting in 0.5 million reads per single-cell.
[SCRB-seq library construction protocol]  The original SCRB-seq protocol was performed as described by Soumillon and colleagues17. To allow profiling of T cells  we introduced a step of RNA purification before reverse transcription with the use of RNAClean XP (A63987  Beckman Coulter). This was used for all modifications assessed in this paper. The following reagents were used for the SCRB-seq modification described in this article: Triton X-100 (T9284-100ML  Sigma-Aldrich)  Buffer TCL (1031576  Qiagen)  2-Mercaptoethanol (M3148-25ML  Sigma-Aldrich)  3âLNA(G)TSO (IDT) and Betaine solution (B0300-1VL  Sigma-Aldrich). After reverse transcription  the single-cell transcriptomes were amplified without pooling for 20 cycles. The barcoded single-cell amplicons were purified with the use of (0.6x) Agencourt AMPure XP beads and libraries were generated as for Drop-seq. The samples were sequenced on Illumina HiSeq 2500 system at the following conditions - rapid run  paired-end  16 bp read 1  49 bp read 2  single-indexed sequencing resulting in 0.5 million reads per single-cell.

value: spleen

value: gp33-specific T cell receptor transgenic P14 CD8 T cells

tag: time

tag: infection

value: acute LCMV Armstrong infection

tag: sample group

value: activated P14 T cells

tag: replicate


title: tSCRB_MM_D8_arm_9

source_name: tSCRB-seq P14s from day 8 acute infection 9

tag: cell type


title: MaelM391Df-Ovary-Brm-input

city: Tokyo

first: Kaoru

last: Sato

address: 35-2 Sakae-cho, Itabashi-ku

country: Japan

institute: Tokyo Metropolitan Institute of Gerontology

data_processing:
mapped to dm6 transcriptome by bowtie2 v2.2.4
culculating FPKM
Genome_build: Drosophila melanogaster dm6
Supplementary_files_format_and_content: bedGraph

source_name: Drosophila ovary

growth_protocol: The flies were maintained at 26°C.

extract_protocol: DNA libraries were raised using NEBNext UltraⅡFS DNA Library Prep Kit (NEB) according to the manufacturer’s instructions.

treatment_protocol: DNA was cross-linked with protein of interest using formaldehyde. After immunoprecipitation using the antibody against the protein of interest, DNA was purified by phenol:chloroform extraction.

tag: tissue

value: ovary

tag: genotype/variation

tag: chip antibody

tag: provider/vender

tag: memo


title: MaelM391Df-Ovary-Brm-ChIP

value: anti-Brm

value: gift from Susumu Hirose in NIG, Japan.


title: DA5.2_ESDM_Rep2

description:
ESDM technical replicate 2
Gene expression data from embryonic stem cell derived macrophages


title: tSCRB_MM_D8_arm_8

source_name: tSCRB-seq P14s from day 8 acute infection 8


title: WT-Ovary-Brm-ChIP

value: WT


title: piwiKD-Osa-input

description:
2 days knockdown
Brm-ChIPseq-processed data file.csv

data_processing:
mapped to dm6 transcriptome by bowtie2 v2.2.4
culculating FPKM
Genome_build: Drosophila melanogaster dm6
Supplementary_files_format_and_content: Brm-ChIPseq-processed data file.csv (file includes FPKM values for each sample)

source_name: OSC

growth_protocol: OSCs were grown at 26°C in culture medium prepared from Shields and Sang M3 Insect Medium (Sigma) supplemented with 0.6 mg/mL glutathione, 10% FBS, 10 mU/mL insulin, and 10% fly extract (Saito et al. 2009).

extract_protocol: DNA libraries were raised using SMARTer ThruPLEX DNA-seq Kit (Clontech) according to the manufacturer’s instructions.

value: Drosophila ovarian somatic cell

value: piwi 2days knockdown


title: tSCRB_MM_D8_arm_6

source_name: tSCRB-seq P14s from day 8 acute infection 6


title: WT-Ovary-Brm-input


title: ctrl-Osa-ChIP

value: egfp 2days knockdown

value: anti-Osa (Developmental Studies Hybridoma Bank, DSHB Cat# osa)


title: tSCRB_MM_D8_arm_4

source_name: tSCRB-seq P14s from day 8 acute infection 4


title: tSCRB_MM_D8_arm_3

source_name: tSCRB-seq P14s from day 8 acute infection 3


title: tSCRB_MM_D8_arm_2

source_name: tSCRB-seq P14s from day 8 acute infection 2


title: ctrl-Osa-input


title: piwiKD-Polybromo-ChIP

value: anti-Polybromo (gift from Susumu Hirose in NIG, Japan; 10.1242/dev.083246)


title: piwiKD-Brm-ChIP

value: anti-Brm (gift from Susumu Hirose in NIG, Japan; 10.1242/dev.083246)


title: ctrl-Polybromo-ChIP


title: piwiKD-Brm-PB-input


title: siBrm_D2 (RNA-Seq)

description: 2 days knockdown

data_processing:
adaptors were trimmed in BGI
depletion of dm6 ribosomal RNAs by bowtie2 v2.2.4
mapped to dm6 transcriptome by bowtie2 v2.2.4
culculating FPKM
Genome_build: Drosophila melanogaster dm6
Supplementary_files_format_and_content: 171220-Piwi-Brm transcripts.xlsx (file includes FPKM values for each sample)

extract_protocol: RNA libraries were raised using TruSeq RNA Library Prep Kit (illunima) according to the manufacturer’s instructions in BGI.

treatment_protocol: Total RNAs were isolated using ISOGEN (Nippon Gene) according to the manufacturer’s instructions.

value: brm 2days knockdown


title: tSCRB_MM_D0_ctrl_3

source_name: tSCBR-seq naÃ¯ve P14s 3

value: non-infected

value: naive P14 T cells


title: tSCRB_MM_D8_arm_1

source_name: tSCRB-seq P14s from day 8 acute infection 1

value: spleen


title: siMael_D2 (RNA-Seq)

description: 2 days knockdown

value: mael 2days knockdown


title: tSCRB_MM_D0_ctrl_2

value: gp33-specific T cell receptor transgenic P14 CD8 T cells


title: A549 10k-RNA

city: San Francisco

middle: Gregory

last: Jones

state: CA

institute: UCSF

description: RNA library of M10k

data_processing:
Library strategy: scRNA-seq
10x cellranger v2.0.1
Target Site analysis with Cassiopeia
Phylogenetic inference with Cassiopeia
Genome_build: "dummy" build, hg19, and GRCh38
Supplementary_files_format_and_content: Target-Site FASTQs were processed with 10X cell ranger v2.0.1. using a "dummy" transcriptome build. Cassiopeia preprocess pipeline was then applied to the resulting possorted_genome_bam to produce allele tables for each mouse, containing Target-Site information for each cell in the Target-Site library. Cassiopeia was then used to create character matrices and phylogenetic trees for each clonal population; trees are stored in newick format. RNA-seq FASTQs were processed with 10X cell ranger v2.0.1.; samples corresponding to M10K and M100k used the hg19 genome build, while M5k and M30k used the GRCh38 genome build. Sparse expression matrices of raw counts, gene names, and cell barcodes are provided as processed files resulting from the 10X processing.

source_name: A549 lu+C31ng adenocarcinoma cell line

growth_protocol: A549 cells (human lung adenocarcinoma line, ATCC CCL-185) were maintained in Dulbecco’s modified eagle medium (DMEM, Gibco) supplemented with 10% FBS (VWR Life Science Seradigm), 2 mM glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin. Cells were transplanted in a matrigel plug into the left lung of immunodeficient mice and grown as xenografts for ~2 months.

extract_protocol:
Cells were washed with PBS with 0.04% w/v bovine serum albumin (BSA, New England Biolabs), filtered through 40 µm FlowMi filter tips filter tips (Bel-Art), and counted according to the 10x Genomics protocol. Approximately 14,000 cells per sample were loaded into the 10x Genomics Chromium Single Cell 3' Library and Gel Bead Kit v2 or v3 kit, and cDNA was reverse-transcribed, amplified, and purified according to the manufacturer's protocol. Resulting cDNA libraries were quantified by BioAnalyzer, yielidng the expected size distribution described in the manufacturer's protocol.
To prepare the Target Site amplicon sequencing library, resulting amplified cDNA libraries were further amplified with custom, Target Site-specific primers containing P5/P7 Illumina adapters and sample indices. PCR amplification was performed using Kapa HiFi HotStart ReadyMix according to the following program: melting at 95C for 3 minutes, then 14 cycles at 98C for 15 seconds and 70C for 20 seconds. Approximately 12 fmol of template cDNA were used per reaction; amplification was performed in quadruplicate to avoid PCR-induced library biases, such as jack-potting. PCR products were re-pooled and purified by SPRI bead selection at 0.9x ratio and quantified by BioAnalyzer.

treatment_protocol: To construct the lineage tracing-competent cell line, A549 cells were transduced by serial lentiviral infection with the three lineage tracing components: (1) Cas9, (2) Target Site, and (3) triple-sgRNAs. First, A549 cells were transduced by Cas9 (mCherry) lentivirus and mCherry+ cells were selected to purity by fluorescenceactivated cell sorting on the BD FACS Aria II. Second, A549-Cas9 cells were transduced by concentrated Target Site (GFP) lentivirus and GFP+ cells were selected by FACS; after sorting, Target Site infection and sorting were repeated two more times for a total of three serial lentiviral transfections, sorting for cells with progressively higher GFP signal after each infection. This strategy of serial transfection with concentrated lentivirus yielded cells with high copy numbers of the Target Site, which were confirmed by quantitative PCR. Third, A549 cells with Cas9 and Target Site were transduced by titered triple-sgRNA (BFP-PuroR) lentivirus and selected

tag: cell line

value: lung adenocarcinoma

tag: plate


title: A549 100k-RNA

description: RNA library of M100k


title: tSCRB_MM_D0_ctrl_1

source_name: tSCBR-seq naÃ¯ve P14s 1


title: A549 LM0-RNA

description: RNA library of preimplantation cells (Day0)


title: siPiwi_D2 (RNA-Seq)

description: 2 days knockdown


title: A549 30k-TS

description: TargetSite library of M30k

source_name: A549 lung adenocarcinoma cell line


title: tSCRB_MM_D8_arm_7

source_name: tSCRB-seq P14s from day 8 acute infection 7

value: acute LCMV Armstrong infection

value: activated P14 T cells


title: piwiKD-Osa-ChIP


title: tSCRB_MM_D8_arm_5

source_name: tSCRB-seq P14s from day 8 acute infection 5


title: siEGFP_D2 (RNA-Seq)

description: 2 days knockdown


title: A549 5k-TS

description: TargetSite library of M5k


title: SCRB_MM_D0_ctrl_5

source_name: Optmization SCBR-seq Triton X100

extract_protocol:
the single-cell transcriptomes are generated with a modified version of the SCRB-seq protocol using Triton X100 for cell lysis
[Purification of mouse and human PBMCs]  Mouse and human lymphocytes were isolated from a mouse spleen and human blood with the use of Lympholyte M (Cedarlane) and Ficoll-Paque PLUS (GE Healthcare) density gradient media respectively. Cells were washed with media and sorted on BD FACS Fusion (100-micron nozzle  standard operation settings  single-cell purity)  where individual cells meeting the gating strategy were sorted in a tube containing media and used immediately for generating Drop-seq single-cell transcriptomes.
[Purification of naive P14 T cells]  Single cell splenocyte suspensions were obtained by mashing total spleens through a 100 Î¼m nylon cell strainer (BD Falcon) and lysing red blood cells with a hypotonic ACK buffer. NaÃ¯ve transgenic P14 CD8 T-cells were isolated using the mouse CD8+ T-cell enrichment kit (Miltenyi Biotech  Bergisch-Gladbach  Germany). Surface staining was performed for 40 min at 4Â°C in supplemented DMEM media (Gibco  ThermoFisher Scientific) using the following antibodies: anti-CD8a (53-6.7  eBioscience)  CD4 (RM4-4  Biolegend)  and VÎ±2 (V20.1  eBioscience). Cells were washed twice with media and sorted on BD FACS Fusion (100-micron nozzle  standard operation settings  single-cell purity). Individual cells meeting the gating strategy were sorted in tube containing media (Drop-seq) and used immediately or directly in lysis buffer into individual wells of a low-binding PCR plate (SCRB-seq)  which was subsequently spun down  snap-frozen on dry ice and stored at -80oC until use. All data were analyzed using FlowJo (TreeStar).
[Purification of activated P14 T cells]  Single cell splenocyte suspensions were obtained by mashing total spleens through a 100 Î¼m nylon cell strainer (BD Falcon) and red blood cells were lysed with a hypotonic ACK buffer. NaÃ¯ve transgenic P14 CD8 T-cells were isolated using the mouse CD8+ T-cell enrichment kit (Miltenyi Biotech  Bergisch-Gladbach  Germany). 2x103 CD45.1+ P14 TCRÎ±Î² were transferred into naÃ¯ve CD45.2+ C57BL/6 mice  followed by their infection. On the day of the sort  single cell splenocyte suspensions were obtained as described above. Activated transgenic P14 CD8 T-cells were isolated using anti-CD45.1 biotin/anti-biotin conjugated microbeads and magnetic MACS cell separation (Miltenyi Biotech  Bergisch-Gladbach  Germany). Surface staining was performed for 40 min at 4Â°C in supplemented DMEM media (Gibco  ThermoFisher Scientific) using the following antibodies: anti-CD8a (53-6.7  eBbioscience)  CD4 (RM4-4  Biolegend)  CD45.1 (A20  eBioscience) and CD45.2 (104  eBioscience). Cells were washed twice with media and sorted on BD FACS Fusion (100-micron nozzle  standard operation settings  single-cell purity). Individual cells meeting the gating strategy were sorted in tube containing media (Drop-seq) and used immediately or directly in lysis buffer into individual wells of a low-binding PCR plate (SCRB-seq)  which was subsequently spun down  snap-frozen on dry ice and stored at -80oC until use. All data were analyzed using FlowJo (TreeStar).
[Drop-seq library construction protocol]  The original Drop-seq protocol was performed as described by Macosko and colleagues16. All runs were performed at 3000 Âµl/hour cell and bead rates with the use of Drop-seq PDMS device (Nanoshift). The following reagents were used for the Drop-seq modification described in this article: Lithium dodecyl sulfate (L9781  Sigma-Aldrich)  Lithium chloride (62476  SigmalAldrich)  Igepal (I8896-50ML  Sigma-Aldrich)  NaCl (S3014  Sigma-Aldrich)  3âLNA(G)TSO (IDT)  Maxima H Minus RT (EP0753  ThermoFisher)  SuperScript IV RT (18090010  ThermoFisher)  SMARTScribe RT (639536  Takara Bio) and Ficoll PM 400 (GE17-0300-10  GE Healthcare). After RNA purification and reverse transcription  the generated single-cell cDNA was amplified for 18 cycles. The barcoded single-cell amplicons were purified with the use of (0.6x) Agencourt AMPure XP beads. 1 ng of the resulting amplified cDNA were used for library preparation with the Illumina Nextera XT DNA Library reagents (FC-131-1024  Illumina). After PCR amplification of the fragmented libraries  the samples were purified with (0.6x) Agencourt AMPure XP beads and eluted in 10 Âµl of molecular grade water. The quality of the resulting library was assessed with the use of Agilent High Sensitivity DNA Kit (5067-4626  Agilent). The library quantification was performed based on the Illumina recommendations (SY-930-1010  Illumina) with the use of KAPA SYBR FAST qPCR Master Mix (KK4600  Kapa Biosystems). The samples were sequenced on Illumina HiSeq 2500 system at the following conditions - rapid run  paired-end  20 bp read 1  45 bp read 2  single-indexed sequencing resulting in 0.5 million reads per single-cell.
[SCRB-seq library construction protocol]  The original SCRB-seq protocol was performed as described by Soumillon and colleagues17. To allow profiling of T cells  we introduced a step of RNA purification before reverse transcription with the use of RNAClean XP (A63987  Beckman Coulter). This was used for all modifications assessed in this paper. The following reagents were used for the SCRB-seq modification described in this article: Triton X-100 (T9284-100ML  Sigma-Aldrich)  Buffer TCL (1031576  Qiagen)  2-Mercaptoethanol (M3148-25ML  Sigma-Aldrich)  3âLNA(G)TSO (IDT) and Betaine solution (B0300-1VL  Sigma-Aldrich). After reverse transcription  the single-cell transcriptomes were amplified without pooling for 20 cycles. The barcoded single-cell amplicons were purified with the use of (0.6x) Agencourt AMPure XP beads and libraries were generated as for Drop-seq. The samples were sequenced on Illumina HiSeq 2500 system at the following conditions - rapid run  paired-end  16 bp read 1  49 bp read 2  single-indexed sequencing resulting in 0.5 million reads per single-cell.


title: DS_mix_HS/MM_D0_ctrl_2

organism:
Homo sapiens
Mus musculus

source_name: Original Drop-seq Human-Mouse Lymphocytes 2

extract_protocol:
the single-cell transcriptomes are generated with the baseline Drop-seq
[Purification of mouse and human PBMCs]  Mouse and human lymphocytes were isolated from a mouse spleen and human blood with the use of Lympholyte M (Cedarlane) and Ficoll-Paque PLUS (GE Healthcare) density gradient media respectively. Cells were washed with media and sorted on BD FACS Fusion (100-micron nozzle  standard operation settings  single-cell purity)  where individual cells meeting the gating strategy were sorted in a tube containing media and used immediately for generating Drop-seq single-cell transcriptomes.
[Purification of naive P14 T cells]  Single cell splenocyte suspensions were obtained by mashing total spleens through a 100 Î¼m nylon cell strainer (BD Falcon) and lysing red blood cells with a hypotonic ACK buffer. NaÃ¯ve transgenic P14 CD8 T-cells were isolated using the mouse CD8+ T-cell enrichment kit (Miltenyi Biotech  Bergisch-Gladbach  Germany). Surface staining was performed for 40 min at 4Â°C in supplemented DMEM media (Gibco  ThermoFisher Scientific) using the following antibodies: anti-CD8a (53-6.7  eBioscience)  CD4 (RM4-4  Biolegend)  and VÎ±2 (V20.1  eBioscience). Cells were washed twice with media and sorted on BD FACS Fusion (100-micron nozzle  standard operation settings  single-cell purity). Individual cells meeting the gating strategy were sorted in tube containing media (Drop-seq) and used immediately or directly in lysis buffer into individual wells of a low-binding PCR plate (SCRB-seq)  which was subsequently spun down  snap-frozen on dry ice and stored at -80oC until use. All data were analyzed using FlowJo (TreeStar).
[Purification of activated P14 T cells]  Single cell splenocyte suspensions were obtained by mashing total spleens through a 100 Î¼m nylon cell strainer (BD Falcon) and red blood cells were lysed with a hypotonic ACK buffer. NaÃ¯ve transgenic P14 CD8 T-cells were isolated using the mouse CD8+ T-cell enrichment kit (Miltenyi Biotech  Bergisch-Gladbach  Germany). 2x103 CD45.1+ P14 TCRÎ±Î² were transferred into naÃ¯ve CD45.2+ C57BL/6 mice  followed by their infection. On the day of the sort  single cell splenocyte suspensions were obtained as described above. Activated transgenic P14 CD8 T-cells were isolated using anti-CD45.1 biotin/anti-biotin conjugated microbeads and magnetic MACS cell separation (Miltenyi Biotech  Bergisch-Gladbach  Germany). Surface staining was performed for 40 min at 4Â°C in supplemented DMEM media (Gibco  ThermoFisher Scientific) using the following antibodies: anti-CD8a (53-6.7  eBbioscience)  CD4 (RM4-4  Biolegend)  CD45.1 (A20  eBioscience) and CD45.2 (104  eBioscience). Cells were washed twice with media and sorted on BD FACS Fusion (100-micron nozzle  standard operation settings  single-cell purity). Individual cells meeting the gating strategy were sorted in tube containing media (Drop-seq) and used immediately or directly in lysis buffer into individual wells of a low-binding PCR plate (SCRB-seq)  which was subsequently spun down  snap-frozen on dry ice and stored at -80oC until use. All data were analyzed using FlowJo (TreeStar).
[Drop-seq library construction protocol]  The original Drop-seq protocol was performed as described by Macosko and colleagues16. All runs were performed at 3000 Âµl/hour cell and bead rates with the use of Drop-seq PDMS device (Nanoshift). The following reagents were used for the Drop-seq modification described in this article: Lithium dodecyl sulfate (L9781  Sigma-Aldrich)  Lithium chloride (62476  SigmalAldrich)  Igepal (I8896-50ML  Sigma-Aldrich)  NaCl (S3014  Sigma-Aldrich)  3âLNA(G)TSO (IDT)  Maxima H Minus RT (EP0753  ThermoFisher)  SuperScript IV RT (18090010  ThermoFisher)  SMARTScribe RT (639536  Takara Bio) and Ficoll PM 400 (GE17-0300-10  GE Healthcare). After RNA purification and reverse transcription  the generated single-cell cDNA was amplified for 18 cycles. The barcoded single-cell amplicons were purified with the use of (0.6x) Agencourt AMPure XP beads. 1 ng of the resulting amplified cDNA were used for library preparation with the Illumina Nextera XT DNA Library reagents (FC-131-1024  Illumina). After PCR amplification of the fragmented libraries  the samples were purified with (0.6x) Agencourt AMPure XP beads and eluted in 10 Âµl of molecular grade water. The quality of the resulting library was assessed with the use of Agilent High Sensitivity DNA Kit (5067-4626  Agilent). The library quantification was performed based on the Illumina recommendations (SY-930-1010  Illumina) with the use of KAPA SYBR FAST qPCR Master Mix (KK4600  Kapa Biosystems). The samples were sequenced on Illumina HiSeq 2500 system at the following conditions - rapid run  paired-end  20 bp read 1  45 bp read 2  single-indexed sequencing resulting in 0.5 million reads per single-cell.
[SCRB-seq library construction protocol]  The original SCRB-seq protocol was performed as described by Soumillon and colleagues17. To allow profiling of T cells  we introduced a step of RNA purification before reverse transcription with the use of RNAClean XP (A63987  Beckman Coulter). This was used for all modifications assessed in this paper. The following reagents were used for the SCRB-seq modification described in this article: Triton X-100 (T9284-100ML  Sigma-Aldrich)  Buffer TCL (1031576  Qiagen)  2-Mercaptoethanol (M3148-25ML  Sigma-Aldrich)  3âLNA(G)TSO (IDT) and Betaine solution (B0300-1VL  Sigma-Aldrich). After reverse transcription  the single-cell transcriptomes were amplified without pooling for 20 cycles. The barcoded single-cell amplicons were purified with the use of (0.6x) Agencourt AMPure XP beads and libraries were generated as for Drop-seq. The samples were sequenced on Illumina HiSeq 2500 system at the following conditions - rapid run  paired-end  16 bp read 1  49 bp read 2  single-indexed sequencing resulting in 0.5 million reads per single-cell.

value: blood

value: peripheral blood mononuclear cells

value: human-mouse mixing experiment


title: SCRB_MM_D0_ctrl_4

source_name: Optmization SCBR-seq Triton X100 + LNA

extract_protocol:
the single-cell transcriptomes are generated with a modified version of the SCRB-seq protocol using Triton X100 for cell lysis and a template switching oligo with locked nucleic acid
[Purification of mouse and human PBMCs]  Mouse and human lymphocytes were isolated from a mouse spleen and human blood with the use of Lympholyte M (Cedarlane) and Ficoll-Paque PLUS (GE Healthcare) density gradient media respectively. Cells were washed with media and sorted on BD FACS Fusion (100-micron nozzle  standard operation settings  single-cell purity)  where individual cells meeting the gating strategy were sorted in a tube containing media and used immediately for generating Drop-seq single-cell transcriptomes.
[Purification of naive P14 T cells]  Single cell splenocyte suspensions were obtained by mashing total spleens through a 100 Î¼m nylon cell strainer (BD Falcon) and lysing red blood cells with a hypotonic ACK buffer. NaÃ¯ve transgenic P14 CD8 T-cells were isolated using the mouse CD8+ T-cell enrichment kit (Miltenyi Biotech  Bergisch-Gladbach  Germany). Surface staining was performed for 40 min at 4Â°C in supplemented DMEM media (Gibco  ThermoFisher Scientific) using the following antibodies: anti-CD8a (53-6.7  eBioscience)  CD4 (RM4-4  Biolegend)  and VÎ±2 (V20.1  eBioscience). Cells were washed twice with media and sorted on BD FACS Fusion (100-micron nozzle  standard operation settings  single-cell purity). Individual cells meeting the gating strategy were sorted in tube containing media (Drop-seq) and used immediately or directly in lysis buffer into individual wells of a low-binding PCR plate (SCRB-seq)  which was subsequently spun down  snap-frozen on dry ice and stored at -80oC until use. All data were analyzed using FlowJo (TreeStar).
[Purification of activated P14 T cells]  Single cell splenocyte suspensions were obtained by mashing total spleens through a 100 Î¼m nylon cell strainer (BD Falcon) and red blood cells were lysed with a hypotonic ACK buffer. NaÃ¯ve transgenic P14 CD8 T-cells were isolated using the mouse CD8+ T-cell enrichment kit (Miltenyi Biotech  Bergisch-Gladbach  Germany). 2x103 CD45.1+ P14 TCRÎ±Î² were transferred into naÃ¯ve CD45.2+ C57BL/6 mice  followed by their infection. On the day of the sort  single cell splenocyte suspensions were obtained as described above. Activated transgenic P14 CD8 T-cells were isolated using anti-CD45.1 biotin/anti-biotin conjugated microbeads and magnetic MACS cell separation (Miltenyi Biotech  Bergisch-Gladbach  Germany). Surface staining was performed for 40 min at 4Â°C in supplemented DMEM media (Gibco  ThermoFisher Scientific) using the following antibodies: anti-CD8a (53-6.7  eBbioscience)  CD4 (RM4-4  Biolegend)  CD45.1 (A20  eBioscience) and CD45.2 (104  eBioscience). Cells were washed twice with media and sorted on BD FACS Fusion (100-micron nozzle  standard operation settings  single-cell purity). Individual cells meeting the gating strategy were sorted in tube containing media (Drop-seq) and used immediately or directly in lysis buffer into individual wells of a low-binding PCR plate (SCRB-seq)  which was subsequently spun down  snap-frozen on dry ice and stored at -80oC until use. All data were analyzed using FlowJo (TreeStar).
[Drop-seq library construction protocol]  The original Drop-seq protocol was performed as described by Macosko and colleagues16. All runs were performed at 3000 Âµl/hour cell and bead rates with the use of Drop-seq PDMS device (Nanoshift). The following reagents were used for the Drop-seq modification described in this article: Lithium dodecyl sulfate (L9781  Sigma-Aldrich)  Lithium chloride (62476  SigmalAldrich)  Igepal (I8896-50ML  Sigma-Aldrich)  NaCl (S3014  Sigma-Aldrich)  3âLNA(G)TSO (IDT)  Maxima H Minus RT (EP0753  ThermoFisher)  SuperScript IV RT (18090010  ThermoFisher)  SMARTScribe RT (639536  Takara Bio) and Ficoll PM 400 (GE17-0300-10  GE Healthcare). After RNA purification and reverse transcription  the generated single-cell cDNA was amplified for 18 cycles. The barcoded single-cell amplicons were purified with the use of (0.6x) Agencourt AMPure XP beads. 1 ng of the resulting amplified cDNA were used for library preparation with the Illumina Nextera XT DNA Library reagents (FC-131-1024  Illumina). After PCR amplification of the fragmented libraries  the samples were purified with (0.6x) Agencourt AMPure XP beads and eluted in 10 Âµl of molecular grade water. The quality of the resulting library was assessed with the use of Agilent High Sensitivity DNA Kit (5067-4626  Agilent). The library quantification was performed based on the Illumina recommendations (SY-930-1010  Illumina) with the use of KAPA SYBR FAST qPCR Master Mix (KK4600  Kapa Biosystems). The samples were sequenced on Illumina HiSeq 2500 system at the following conditions - rapid run  paired-end  20 bp read 1  45 bp read 2  single-indexed sequencing resulting in 0.5 million reads per single-cell.
[SCRB-seq library construction protocol]  The original SCRB-seq protocol was performed as described by Soumillon and colleagues17. To allow profiling of T cells  we introduced a step of RNA purification before reverse transcription with the use of RNAClean XP (A63987  Beckman Coulter). This was used for all modifications assessed in this paper. The following reagents were used for the SCRB-seq modification described in this article: Triton X-100 (T9284-100ML  Sigma-Aldrich)  Buffer TCL (1031576  Qiagen)  2-Mercaptoethanol (M3148-25ML  Sigma-Aldrich)  3âLNA(G)TSO (IDT) and Betaine solution (B0300-1VL  Sigma-Aldrich). After reverse transcription  the single-cell transcriptomes were amplified without pooling for 20 cycles. The barcoded single-cell amplicons were purified with the use of (0.6x) Agencourt AMPure XP beads and libraries were generated as for Drop-seq. The samples were sequenced on Illumina HiSeq 2500 system at the following conditions - rapid run  paired-end  16 bp read 1  49 bp read 2  single-indexed sequencing resulting in 0.5 million reads per single-cell.


title: DS_mix_HS/MM_D0_ctrl_3

source_name: Original Drop-seq Human-Mouse Lymphocytes 3


title: ctrl-Brm-PB-input

value: Drosophila ovarian somatic cell


title: A549 100k-TS

description: TargetSite library of M100k


title: DS_mix_HS/MM_D0_ctrl_1

source_name: Original Drop-seq Human-Mouse Lymphocytes 1


title: ctrl-Brm-ChIP

source_name: OSC

value: egfp 2days knockdown