title: Illumina NovaSeq 6000 paired end sequencing; Time-resolved single-cell analysis of Brca1 associated mammary tumorigenesis reveals aberrant differentiation of luminal progenitors
library_construction_protocol: 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.