Illumina NovaSeq 6000 paired end sequencing; Time-resolved single-cell analysis of Brca1 associated mammary tumorigenesis reveals aberrant differentiation of luminal progenitors
Time-resolved single-cell analysis of Brca1 associated mammary tumorigenesis reveals aberrant differentiation of luminal progenitors
All primary human breast tissue was derived from women undergoing reduction mammoplasties with no known genetic history (n = 4) and prophylactic mastectomies from women with germline BRCA1 mutations (n = 4) obtained from the Breast Cancer Now Tissue bank (15/EE/0192) Sorted cells were spun down and resuspended in RLT, and RNA was extracted using the RNeasy micro kit (Qiagen) according to manufacturer instructions. DNA was degraded by adding 20U Rnase-free DnaseI (Roche) for 30 min at room temperature. DnaseI treatment was performed on columns.    RNA  samples  were  QC'd  with  Qubit  and  Tapestation.  3 ug  of  total  RNA  was  next used  in  the  NEBNext®  Poly(A)  mRNA  Magnetic  Isolation Module (E7490). Next, 30 ng of the PolyA RNA was used with the NEBNext Ultra II Directional  RNA  Library  Prep  Kit  for  Illumina  (E7760),  with  8  cycles  of  PCR.  QC  of  the  finished libraries was done with Qubit and Tapestation.