Depletion of S100A4+ stromal cells results in abnormal nipple development and nursing failure
Depletion of S100A4+ stromal cells results in abnormal nipple development and nursing failure
Thoracic and inguinal nipples were dissected from the skin of L1 lactating dams as a patch of a approx. 2 mm in diameter and transferred to QIAzol Lysis Reagent (Qiagen). All samples were stored in -80 °C until collection of the complete experimental sample set. RNA isolation from frozen tissue was performed using 1.4mm Ceramic Bead Media (Revity), QIAzol Lysis Reagent (Qiagen) and NucleoSpin RNA kit (Macherey-Nagel). First, ceramic beads were added to the tissue in 800 μl of Qiazol followed by tissue disruption in homogenizer Bead Ruptor 4 (Omni International). Subsequently, the aqueous phase containing RNA was separated using 80 μl of BCP (Molecular Research Center). The aqueous phase was pipetted out, mixed with 70% EtOH in a ratio of 1:1 and applied to the column from NucleoSpin RNA kit. The rest of the isolation was performed according to manufacturer's protocol of NucleoSpin RNA kit. RNA concentration was determined by Nanodrop 2000c (Thermo Scientific) and RNA integrity was checked on the Fragment Analyzer using RNA Kit 15 nt (Agilent Technologies). 500 ng of total RNA was used as input for library preparation using QuantSeq 3′ mRNA-Seq V2 Library Prep Kit FWD with UDI 12 nt (Lexogen) in combination with UMI Second Strand Synthesis Module for QuantSeq FWD (Lexogen). Library quantity and size distribution was checked using QuantiFluor dsDNA System (Promega) and High Sensitivity NGS Fragment Analysis Kit (Agilent Technologies).