title: GSM4832641: msLung_4Male/Castrated_12wks_nuc; Mus musculus; RNA-Seq
library_construction_protocol: All buffers used were pre-chilled to 4oC and all steps were performed on ice or at 4oC, unless otherwise mentioned. Perfused mouse lungs, transported in RPMI (phenol-red free) at 4oC, were washed 2x with 1x PBS. Excess buffer from the lungs were wicked away, lungs were transferred to individual tubes and then the tubes were flash frozen in liquid nitrogen and used immediately or stored at -80oC. Tissues were then minced with a fresh, RNase free scalpel and added to a tube congaing chilled nuclei isolation buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.16% Nonidet™ P40 and 0.2 U/µL RNase inhibitor) at a tissue : buffer ratio of 0.1g : 100 µL. After incubating at 4oC with rotation for 10 min, tubes were incubated at 4oC for 2 min to let the debris settle down, and the supernatant containing the nuclei were collected. The supernatant was centrifuge at 500xg, 4oC for 5 min to pellet the nuclei. The supernatant was discarded and the pellet was resuspended in 500 µL resuspension buffer (1x PBS, 0.4% BSA and 0.2 U/µl of RNase inhibitor) and passed through a 70 µm filter. The above step was repeated, but the resuspended nuclei were passed through a 40 µm filter, and this step was repeated one more time. The nuclei suspension was centrifuged at 500xg, 4oC for 5 min to pellet the nuclei and the nuclei were resuspended in 100 µL of resuspension buffer. After counting (tryphan blue staining of ~10 µL of the sample), the nuclei suspension was diluted to a concentration of 1 million nuclei/mL and immediately used for 10x barcoding. Approximately 5,000 nuclei from each sample were captured in Gel Bead-In-EMulsions (GEMs) containing ~ 750,000 unique molecular Indexes (UMIs) using the 10X Chromium Controller module. The GEMs were generated in a chip, containing Master Mix/cells mixture, Gel Beads and partitioning oil (placed in distinct compartments). Subsequent to dissolution of the Single Cell 3' Gel Bead in a GEM, primers containing an Illumina P7 and R2 sequence (read 2 sequencing primer), a 14 bp 10x barcode, a 10 bp randomer and a poly-dT sequence werere released and mixed with cell lysate and reaction master mix. Reverse transcription produced barcoded, full-length cDNA. After GEMs were broken and cDNA cleaned using Solid Phase Reversible Immobilization (SPRI) beads the contents were subject to PCR. The sheared (Covaris) amplified cDNA were subjected to end repair, A-tailing, adaptor ligation, and PCR. Samples were sequenced on Illumina HiSeq.