2024-07-13
ENA first public
ERC000011
ENA-CHECKLIST
Harvard T.H. Chan School of Public Health
INSDC center name
public
INSDC status
ERS20445921
SRA accession
E-MTAB-14214:Sample 2
Submitter Id
17
day
age
ArrayExpress
broker name
placenta
cell_type
not collected
collection date
house mouse
common name
Protocols: To prepare single cell suspension for scRNA seq, fresh whole mouse placentae were washed twice with cold PBS and were enzymatically digested in Liberase TL (Roche, 1:10 in PBS) for 15 min at 37ºC with shaking. Then, placentae were manually disaggregated by pulling up and down 10 times on a 3-mL syringe carrying a 16G needle, placed back at 37ºC with shaking for another 15 minutes, then manually disaggregated again with an 18G needle. The digestion was stopped by adding 2 mL fetal bovine serum (Invitrogen) and was passed through a 70 m cell strainer. Cells were collected by centrifugation at 300xg for 5 minutes and resuspended in 10 mL PBS/0.04% BSA for counting. Erythrocytes were depleted from the samples by MACS using Anti-Ter-119 MicroBeads and LS columns (Miltenyi) according to the MicroBeads’ protocol. We utilized two time-pregnant mice for each treatment group (control and As exposure). From each pregnant mouse, we collected 7-8 placentae corresponding to the number of pups. Cell suspensions were processed to generate single-cell RNA-Seq libraries using Chromium Next GEM Single Cell 3′ GEM, Library and Gel Bead Kit v3.1 (10x Genomics), per the manufacturer’s recommendations, as summarized below. Samples were loaded on a Chromium Single-Cell Instrument (10x Genomics, Pleasanton, CA, USA) to generate single-cell GEMs (Gel Bead-in-Emulsions) (16,500 cells per sample, to target 10,000 cells for capture).  GEM reverse transcription (GEM-RT) was performed to produce a barcoded, full-length cDNA from poly-adenylated mRNA. After incubation, GEMs were broken, the pooled GEM-RT reaction mixtures were recovered, and cDNA was purified with silane magnetic beads (DynaBeads MyOne Silane Beads, ThermoFisher Scientific). The purified cDNA was further amplified by PCR to generate sufficient material for library construction.   Enzymatic fragmentation and size selection was used to optimize the cDNA amplicon size and indexed sequencing libraries were constructed by end repair, A-tailing, adaptor ligation, and PCR. Final libraries contain the P5 and P7 priming sites used in Illumina bridge amplification.
description
fetal
developmental stage
N/A
genotype
not collected
geographic location (country and/or sea)
fetal
individual
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
placenta
organism part
Mus musculus
scientific_name
male
sex
C57BL/6
strain
Sample 2
title