title: Illumina NovaSeq 6000 paired end sequencing; Distinct chromatin signatures in plant male germline cells - scRNA-seq
design: Distinct chromatin signatures in plant male germline cells - scRNA-seq
library_construction_protocol: Open flower buds were freshly collected from pollen marker line into 9% sucrose solution and matured pollen grain were enriched by vortex and filtered through a 60 um nylon mesh. The sperm nuclei and vegetative nuclei were isolated by FACS using a method described previously (cite paper). Briefly, pollen grain were resuspended in ice-cold Galbraith buffer (45mM MgCl2, 30mM Sodium Citrate, 20mM MOPS, 1% Triton X-100, pH to 7.0) supplemented with 1X complete protease inhibitor cocktail (Roche) and pollen wall was then disrupted by additional vortexed at 60Hz for 30sec with glass beads (sigma). The resulting pollen wall debris were removed by filtering through a 10 um nylon mesh. The crude suspension was ready for FACS on a FACSAria III cell sorter. FACS was carried out in standard configuration using 70um nozzle. A 488nm laser was used for GFP excitation and a 561nm laser for RFP excitation.  The pollen marker line (ACT::H2B-mRFP labels vegetative nucleus, MGH3P::MGH3-eGFP labels sperm nucleus in mature Arabidopsis pollen) used in this study was kindly provided by prof. Jorg Dieter Becker (Cite method paper). Plants were grown in climate controlled greenhouse under long day conditions (16hr light, 8hr dark) at 22C.  Amplified cDNA was purified using Qiagen MinElute PCR Purification Kit. Library DNA was purified using AmpureXP beads using 1:1 ratio. Single-cell RNA-seq experiments were performed based on a modified SMART-seq3 method. For sperm and vegetative nuclei scRNA-seq, single nucleus of each cell type was sorted into each well of 384-well PCR plate (Eppendorf, Cat No. :76019-008) that contains 2ul lysis buffer (6.67% PEG, 0.1% Triton X-100, 0.53U/ul RNase inhibitor, 0.67mM/each dNTPs) and 1ul barcoded oligo-dT RT-primers (2 µM). Plates were immediately spun down after sorting and stored at -80 °C. On the day of the experiment, plates were thawed on ice. Then Maxima H- RT enzyme (ThermoFisher, cat no. EP0751) and Template Switching Oligo (TSO) were added to each well to generate first-strand cDNA. Then cDNA was amplified using the KAPA HiFi master mix (KAPA, cat. no. KK2601) with primers annealed to the RT primer and TSO. The resulting amplified single cell cDNA from a plate were pooled together and purified with a Qiagen PCR purification Kit (Cat No.28104).  Eluted cDNA samples were treated with Exo I to remove the excess primers and purified with SPRI beads. cDNA concentration was measured using a Qubit 3 fluorometer, and 50ng purified full-length cDNAs were used for Tn5 transposome based library construction. The library was purified with VATHS DNA purification beads and sequenced on the Illumina platform to generate 150 bp paired-end reads. In total, 601 successfully sorted sperm and vegetative nuclei were analyzed in this study.