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Sample GSM4731908

Query DataSets for GSM4731908

Status

Public on Jul 07, 2022

Title

Trophoblast cells, HS091

Sample type

SRA

Source name

Blastocyst

Organism

Homo sapiens

Characteristics

Stage: Day 9
growth condition: in vitro culture
cell type: trophoblast cells

Treatment protocol

None

Growth protocol

Donated human embryos (at the cleavage or blastocyst stage) were thawed and cultured in 8-well chambers to day 13.

Extracted molecule

total RNA

Extraction protocol

Single cells were isolated from embryos at embryonic days 8, 9, 10, 11, 12, and 13, incubated with TrypLE Express reagent for 10-15 min at 37°C, and dissociated into single cells. Single cells were randomly picked with a mouth pipette in 0.1% BSA and then transferred to 2.5 μl lysis buffer. The cells were transferred into a 0.2 mL PCR tube (Eppendorf) containing cell lysis buffer and kept at −80°C for library preparation.
The linearized RNA molecules were releasein a thermocycler at 72 °C for 3 min at first. Then, transfer the reactions immediately on ice. Add 2.85 µl RT mixture into every reaction with component as SuperScript II reverse transcriptase, RNase Inhibitor, 5× Superscript II first-strand buffer, dithiothreitol (DTT), betaine, MgCl2, and template switch oligo (TSO) primer. Spin down the 5.4 µl samples in the thermocycler at 25 °C for 5 min, 42 °C for 60 min, 50 °C for 30 min, and then 70 °C for 10 min, 4 °C hold. Prepare 7.5 µl PCR mixture (6.25 μL 2× KAPA HiFi HotStart ReadyMix, ISPCR oligo, and 3′ Anchored oligo) to add in every tube reaction, and amplify samples for 19 cycles. Pool together samples with different barcodes (up to 96 barcodes) and purify DNA. Then prepare the second round of amplification with bio-tinylated index primer and ISPCR oligo for an additional five cycles. Subsequently, purify the biotinylated cDNAs further with 0.8× Ampure XP beads and elute cDNA in 50 µl EB. Next, shear the cDNAs into approximately 300-bp fragments with sonicator.Enrich the cDNA fragments by Dynabeads® MyOneTM Streptavi- din C1. Libraries were prepared with KAPA Hyper Prep Kits. Use the NEB U-shaped adapter for ligation. Finally, amplify the adapter-ligated fragments using an Illumina QP2 primer and a short universal primer for 9 cycles.
Single Cell RNA-Seq

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

The paired-end reads Smart-seq2 data was processed using the standard pipeline of Drop-seq_tools-2.0.0 (http://mccarrolllab.com/dropseq/) with modifications.
For read2, bases 1 to 8 were tagged with cell-barcode “XC,”and bases 9 to 16 were tagged with UMI “XM”.
Read1 was trimmed at the 5' end to remove adaptor and TSO sequence, and the 3' end was trimmed to remove poly(A) sequences of length 6 or more and then aligned to hg38 genome reference using STAR aligner.
Gene expression matrix for each cell was then generated with DigitalExpression command.
Genome_build: hg38
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every cell

Submission date

Aug 19, 2020

Last update date

Jul 07, 2022

Contact name

Xiangxiang Jiang

E-mail(s)

jzhongqiang@126.com

Phone

18810332066

Organization name

Institute of Zoology, Chinese Academy of Sciences