Illumina HiSeq 2500 paired end sequencing; GSM3531672: P301_3; Homo sapiens; RNA-Seq

P301_3

We perfomed single-cell RNA-sequnecing of around 10,000 cells from normal human liver tissue to construct a human liver cell atlas. We reveal previously unknown subtypes in different cell type compartments. We also use our normal liver cell atlas to infere perturbed phenoytpes of cells from HCC samples, human cells engrafted into a mouse liver and liver organoids. Overall design: Single cells were isolated from human liver resection speciments and then sorted by FACS into 384 well plates in a unbiased way and on the basis of cell surface markers for distinct cell types. ScRNA-seq was done using the mCelSeq2 protocol cellbarcodes_cellid.csv Supplemetary file contains cellds and one of the 192  unique cellbarcode associated with the cellid.

A Human Liver Cell Atlas reveals Heterogeneity and Epithelial Progenitors

Submitted on 19-JUN-2019

We perfomed single-cell RNA-sequnecing of around 10,000 cells from normal human liver tissue to construct a human liver cell atlas. We reveal previously unknown subtypes in different cell type compartments. We also use our normal liver cell atlas to infer perturbed phenoytpes of cells from HCC samples, human cells engrafted into a mouse liver and liver organoids. Overall design: Single cells were isolated from human liver resection specimens and then sorted by FACS into 384 well plates in a unbiased way and on the basis of cell surface markers for distinct cell types. ScRNA-seq was done using the mCelSeq2 protocol cellbarcodes_cellid.csv Supplemetary file contains cellds and one of the 192  unique cellbarcode associated with the cellid.

Liver specimens were perfused and treated with collagenase followed by Percoll purifcation/clean up steps The amplified RNA (aRNA) was reversely transcribed in order to generate libraries for sequencing. 1 µl of custom random hexamer RT primers (GCCTTGGCACCCGAGAATTCCANNNNNN) and 0.5 µl of 10mM dNTP were added to the aRNA. The samples were incubated at 65˚C for 5 min and cooled to 4˚C afterwards. Thereafter 4 µl of First Strand Reaction Mix containing SuperScript™ II Reverse Transcriptase and Buffer (Invitrogen, 18064014) and RnaseOUT™ (Invitrogen, 10777019) was added and samples were first incubated at 25˚C for 10 min followed by incubation at 42˚C for 1 hour. To every sample 2 µl of one uniquely indexed RPI index primer and 2 µl of RP1 primer (TruSeq Library Prep, sequences available from Illumina), 25 µl of Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB, M0531) and 11 µl of nuclease-free water were added. PCR was performed using 98˚C for 30 s as initial step, followed by 11 cycles of amplification (98˚C for 10 s, 60˚C for 30 s, 72˚C for 30 s) and a final extension at 72˚C for 10 min. To each sample 1 volume (50 µl) of Agencourt® AMPure XP beads (Beckman Coulter, A63881) was added and the samples were incubated at 25˚C for 15 min. The samples were incubated for 5 min on a magnetic stand (96 well format), the supernatant was removed and the beads were washed twice by adding 180 µl of 80 % ethanol and by incubating for 40 s. Ethanol was removed and beads were dried for 10 min at room temperature. DNA was eluted with 25 µl nuclease-free water and the purification was repeated with an adjusted volume of magnetic beads (25 µl). After the final step the samples were eluted in 10 µl nuclease-free water. DNA concentration and fragment size were determined using the Qubit® dsDNA HS Assay Kit (Invitrogen, Q32854) and the Agilent High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies, 5067-4627), respectively. Libraries were sequenced on an Illumina HiSeq 2500 System in high output run mode at a depth of 150000 to 200000 reads per cell. CEL-Seq2 (Hasimshony et al. 2016, Genome Biology, DOI: 10.1186/s13059-016-0938-8)

A Human Liver Cell Atlas reveals Heterogeneity and Epithelial Progenitors

Submitter Supplied Information

Samples