Nature research paper: Developmental dynamics of two bipotent thymic epithelial progenitor types
for 3 min at 4 °C, snap-frozen in liquid nitrogen and stored at −80 °C before further processing. To convert RNA into cDNA, 160 nl of reverse-transcription reaction mix including 0.4 µM template-switch oligonucleotide and 2.2 μl of second-strand reaction mix were added.
For the purpose of simultaneous transcriptome and barcode analysis, the volume of each well was equally split in half; that is, 1.1 µl per well was transferred to a new 384-well plate. The original plate was used for barcode analysis, while the copy of the plate was used for analysis of individual cell transcriptomes.
For barcode analysis, all transcripts in each well were amplified by template-switch PCR and 5′-CAGAGTTCTACAGTCCGA-3′ ), followed by amplification of the scar region oftranscripts , 5′-GCCTTGGCACCCGAGAATTCCATAGCGATGATGAACCAGGTTATGACC-3′ ) using the PrimeSTAR GXL system . Libraries were completed by addition of full-length adaptors by PCR . Libraries from 96 cells were pooled before clean-up.
In total, 32 libraries , 32 libraries , 28, 40 and 80 libraries , 28 libraries , 32 libraries , 44 libraries and 60 libraries were sequenced on the Illumina HiSeq 2500 or NovaSeq 6000 sequencing system at a depth of ~170,000 reads per cell.. The transcriptome contained all gene models based on the mouse ENCODE VM9 release downloaded from the UCSC genome browser, comprising 57,207 isoforms, with 57,114 isoforms mapping to fully annotated chromosomes .
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