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Sample GSM3814116

Query DataSets for GSM3814116

Status

Public on Aug 20, 2020

Title

72406

Sample type

SRA

Source name

chorionic villi

Organism

Homo sapiens

Characteristics

tissue: primary placental tissue
developmental stages: first trimester
fetal sex: female

Extracted molecule

total RNA

Extraction protocol

Fresh placental tissues of 5~10mg were placed in collection medium and transferred to lab and kept at 4°C overnight before cell preparation. The collection medium was αMEM with sodium heparin (100,000 units/100ml H2O, Sigma), gentamycin (0.5%, Invitrogen) and antibiotic-antimycotic (2%, Invitrogen). The tissue was washed in cold PBS to remove maternal contamination and minced with a sterile scalpel, then mixed with 0.25% trypsin, 300U/ml collagenase and 200μg/ml DNAse I and incubated in a 37°C, 5% CO2 incubator for 90 minutes with occasional agitation. After centrifugation at 1200 rpm for 10min, cell pellets were carefully resuspended in Chang medium (Irvine Scientific). To avoid overpopulation of erythrocytes in the captured cell population, 1xRBC lysis buffer was applied to the cell suspension for 15min at room temperature, after which, cells were carefully washed and resuspended in 100μl Chang medium and strained using a 70μm flowmi cell strainer (Bel-Art) immediately before library construction.
Single-cell RNA-seq libraries were prepared per Single Cell 3′ v2 Reagent Kits User Guide (10x Genomics, Pleasanton, California). Single cell suspensions were loaded on a Chromium Controller instrument (10X Genomics) to generate single-cell Gel Bead-In-EMulsions (GEMs). GEM-RT were performed in a Veriti 96-well thermal cycler (Thermo Fisher Scientific, Waltham, MA), following which, GEMs were harvested and the cDNAs were amplified and cleaned up with SPRIselect Reagent Kit. Indexed sequencing libraries were constructed using Chromium Single-Cell 3′ Library Kit for enzymatic fragmentation, end-repair, A-tailing, adapter ligation, ligation cleanup, sample index PCR, and PCR cleanup. The barcoded sequencing libraries were quantified using the KAPA Library Quantification Kit (KAPA Biosystems, Wilmington, MA).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

Cell Ranger 2.0.0 (10X Genomics) was used to demultiplex reads and convert raw base call files into fastq format. Reads alignment was performed by using STAR (version 2.5.1) with hg38 transcriptome reference from Gencode 25 annotation, containing all protein-coding and long non-coding RNA genes. Expression counts for each gene in all samples were collapsed to unique molecular identifier (UMI) counts using Cell Ranger 2.0.0 (10X Genomics).
Genome_build: hg38
Supplementary_files_format_and_content: csv with raw expression matrix for all of six samples

Submission date

May 23, 2019

Last update date

Aug 20, 2020

Contact name

Margareta D Pisarska

E-mail(s)

pisarskam@cshs.org

Phone

3106148307

Organization name

Cedars Sinai Medical Center