title: 10x_2560


title: 10x_2559

status: Public on May 01 2020

type: SRA

city: Rehovot

first: Itay

last: Tirosh

address: Herzl 234

country: Israel

institute: WEIZMANN INSTITUTE OF SCIENCE

library_source: transcriptomic

data_processing:
Paired-end, 38-base reads were mapped to the UCSC hg19 human transcriptome using Bowtie with parameters "-q --phred33-quals -n 1 -e 99999999 -l 25 -I 1 -X 2000 -a -m 15 -S -p 6". Expression values were calculated by   RSEM v1.2.3 in paired-end mode using parameters "--estimate-rspd --paired end -sam -p 6", from which TPM values for each gene  were extracted. For the subset of cells processed with 10X, we used CellRanger with the default parameters.
We examined as quality control the number of genes detected in each cell and conservatively excluded cells with fewer than 2,000 detected genes.
Genome_build: hg19
Supplementary_files_format_and_content: Two tab-delimited files containing log2(TPM/10+1) values (TPM=transcript-per-million reads) of 9609 cells profiled by 10x droplet scRNA-seq (10x file) and of 1297 cell profiled by SmartSeq2 (SS2 file). All quantified 23,686 genes are included in the SS2 file and 11,548 genes detected in at least 10 cells are included in the 10x file. Header lines include Cell ID, patient ID, time point of sample acquisition, and the cells assignment into clusters and coordinates in a tSNE analysis (as shown in an associated manuscript).

molecule: polyA RNA

organism: Homo sapiens

source_name: Malignant ascites  single cell

extract_protocol:
RNA from single cells was first purified with Agencourt RNAClean XP beads (Beckman Coulter)
Libraries from isolated single cells were generated based on the Smart-seq2 protocol (Picelli  2014) with the following modifications.oligo-dT were used to prime reverse transcription with Maxima reverse transcriptase and locked TSO oligonucleotide, which was followed by 20 cycle PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA Biosystems) with subsequent Agencourt AMPure XP bead purification as described. Libraries were tagmented using the Nextera XT Library Prep kit (Illumina) with custom barcode adapters (sequences available upon request). Libraries from 384 cells with unique barcodes were combined and sequenced using a NextSeq 500 sequencer (Illumina). For a subset of samples, single cells were processed through the 10X Chromium 3 Single Cell Platform using the Chromium Single Cell 3’ Library, Gel Bead and Chip Kits (10X Genomics, Pleasanton, CA), following the manufacturer’s protocol. Briefly, cells were added to each channel of a chip to be partitioned into Gel Beads in Emulsion (GEMs) in the Chromium instrument, followed by cell lysis and barcoded reverse transcription of RNA in the droplets. Breaking of the emulsion was followed by amplification, fragmentation and addition of adapter and sample index.

treatment_protocol: Ascites fluid was drained by an interventional radiologist and transferred for further processing in closed vacuum bottles. De-identified patient information, including their ovarian cancer histology, stage, treatment history, and BRCA mutation status were collected. Immediately following drainage, malignant ascites was transported on ice, aliquoted into 50 mL conical tubes (BD Falcon) and spun for 5 min at 580 × g at 4°C. The supernatant was aspirated and the remaining pellet was resuspended with 5 mL of hypotonic lysis buffer ACK (Life technologies) and incubated on ice for 5 min. 20 ml of PBS was used to quench the lysis buffer. Cell suspensions were pooled and pipetted into a new 50 mL conical tube through a 100 µm mesh and spun for 5 min at 580 × g at 4°C. Hypotonic lysis was repeated until no visible red blood cell component was present, usually for a total of 2-3 times (for the plate-based approach). Following red blood cell lysis, the cell pellet was resuspended in PBS with 2% (v/v) FBS. These cells were then used for both plate-based and droplet-based scRNA-seq. For plate-based scRNA-seq, cells were labeled with the following fluorophore-conjugated flow-cytometry antibodies: live/dead stain with Calcein-AM (LifeTechnologies), 7-AAD (7-Aminoactinomycin D) (LifeTechnologies), CD45-FITC (LifeTechnologies), EpCAM-PE (Miltenyi Biotec) and CD24-PE/Cy7 (BioLegend). Cells were incubated for 30 min on ice in the dark. Cells were washed twice by resuspending with PBS with 2% (v/v) FBS and spun 5 min at 580 × g at 4°C. Flow-cytometry and sorting was performed on a BD Biosciences cell sorter (IIu or UV). Following doublet exclusion, Calceinhigh7AAD-CD45-EPCAM+CD24+ single cells were sorted into 96-well microtiter plates (on a plate chiller) that were prepared with 10uL cell lysis buffer (TCL + 1% β-mercaptoethanol). Following completion of cell sorting, plates were covered with aluminum lids, vortexed for 10 seconds, centrifuged for 2 min at 580 × g at 4°C and immediately placed on dry ice prior to storage at -80°C.

tag: cell type

value: Malignant ascites  single cell

tag: patient id

tag: time point


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