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To validate the bioinformatics tools, we randomly sampled a subset of the total data and manually dissected the raw reads one by one to identify the CDR3 sequences.
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The enzymatic digestion with 1 mg/ml (activity 0.199 U/mg) Collagenase B and 0.033 mg/ml Trypsin was performed in oxygenated Tyrode solution with 25 mM BDM and 50 µM CaCl2 for 8 12 min and after manually dissecting the tissue the reaction was stopped in stopping solution (oxygenated Tyrode solution with 25 mM BDM, 50 µM CaCl2 and 5 % FCS).
The calyx abscission zone (AZ) tissues, containing a few layers of AZ cells on the proximal side of the separation line and adjacent cells, were manually dissected from the calyx tube samples, using a razor blade of 1 mm.
The initial paraffin blocks were manually dissected and the selected tumour area was embedded in a new paraffin block.
The maternal basal plate (decidua) was manually dissected from the fetal placental tissue.
Cross sections of stems were manually dissected from the first internode of developmentally equivalent transgenic and control plants.
Regions were manually dissected from the frozen blocks to provide a consistent tumor cell content of more than 70% in tissues used for analysis.
After removal of paraffin wax using graded xylene- and alcohol dilutions following routine protocols, tumor cells were manually dissected from the slides.
This is particularly important for the TE and ICM samples since these were manually dissected and confirms the reproducibility of the dissection.
RNA was obtained from the manually dissected FFPE samples using the same method as for the Australian CRC series.
The remaining periostracum and proteinaceous components of the shell were manually dissected away from the animal with fine dissecting forceps.
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